κB-Ras/RalGAP complexes limit the activity of Ral GTPases, which function in EGFR/Ras signaling. RalGAP expression is down-regulated in pancreatic cancer; however, the role of RalGAP and Ral GTPases in tumor development in vivo remained unclear. Here, we show that pancreatic RalGAPβ deficiency alone is sufficient to induce inflammation and neoplasia in vivo. We identify that this phenotype is triggered by disturbance of the secretory pathway and polarized exocytosis in acinar cells, demonstrating that RalGAP complexes uphold spatial control of Ral activity. We furthermore show that RALGAPβ deficiency results in defective primary cilium assembly, a process required for efficient acinar regeneration upon inflammation. Only primary cilium formation depends on κB-Ras proteins, suggesting that κB-Ras proteins are not essential for all RalGAP complex-controlled processes. In combination with an oncogenic KRAS G12D mutation, RalGAPβ deficiency leads to a dramatic shortening of tumor latency and median survival. Our results highlight an important role of RalGAP/Ral signaling in upholding acinar cell identity and preventing pancreatic cancer development.
© 2025 Apken et al.
Product Citations: 1,024
RalGAP complexes control secretion and primary cilia in pancreatic disease.
In Life Science Alliance on 1 August 2025 by Apken, L. H., Barz, H., et al.
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In Scientific Reports on 3 June 2025 by Lei, R., da Silva, T. B., et al.
Studies have shown that mesenchymal stromal cells (MSCs) could secrete a variety of bioactive particles, including small extracellular vesicles (sEVs), that might be a key intermediate to the beneficial paracrine effects of MSC therapy. In this study, we harvested the conditioned medium (CM) from human bone marrow mesenchymal stromal cells (hBM-MSCs) and normal human dermal fibroblasts (NHDFs), fractionated into the sEV fraction and non-small extracellular vesicle (NsEV) fraction, and compared their functions on NHDF migration and proliferation-key processes in wound healing, coupled with transcriptomic analysis through mRNA sequencing to assess gene expression changes in the recipient NHDFs. Our findings show that sEV, NsEV and CM from hBM-MSCs had an overall promotive effect on migration behaviour of NHDFs, but MSC-sEV surpassed the effects of other MSC secretome fractions and their NHDF counterparts (HDF-sEV). Gene ontology analysis revealed enrichment of pathways related to migration, and significant changes in genes within the regulation of proliferation pathway. Our study provides referential significance for the choice of cellular secretome fractions to be used in wound healing studies and insights into the effects of MSC secretome in the migration and proliferation of healthy fibroblasts, besides their effects on gene expression changes.
© 2025. The Author(s).
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In Bio-protocol on 20 May 2025 by Ishida, K. & Morita, E.
Orthoflavivirus is an enveloped, positive-stranded RNA virus that buds into the endoplasmic reticulum (ER) lumen. The budded virus particles are subsequently transported to the Golgi apparatus and secreted into the extracellular environment via the conventional secretion pathway. In this protocol, we describe a method for monitoring the secretion of Orthoflavivirus particles from the ER. To visualize intracellular membrane trafficking, we combine two distinct imaging techniques: the retention using selective hooks (RUSH) system and the split green fluorescent protein (GFP) system. In this approach, GFP11, a peptide tag fused to prME, the outer coat structural protein of Japanese encephalitis virus particles, was co-expressed in HeLa cells along with two additional components: GFP1-10 fused to a streptavidin-binding peptide and a hook construct consisting of streptavidin fused to the ER retention sequence KDEL. Time-lapse imaging was performed after the addition of biotin, which releases the captured GFP-labeled subviral particles from the ER. This method enables synchronized visualization of intracellular subviral particle trafficking and serves as a valuable tool for analyzing the maturation process of Orthoflavivirus particles within cells. Key features • Synchronized intracellular movement of Orthoflavivirus particles is visualized by the retention using selective hooks (RUSH) system. • Split GFP system is used to label viral particles. • This protocol has broader applications in investigating the transport of secretory proteins, especially those that are challenging to tag with full-length fluorescent proteins.
©Copyright : © 2025 The Authors; This is an open access article under the CC BY-NC license.
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SARS-CoV-2 ORF3a drives dynamic dense body formation for optimal viral infectivity.
In Nature Communications on 12 May 2025 by Hartmann, S., Radochonski, L., et al.
SARS-CoV-2 hijacks multiple organelles for virion assembly, of which the mechanisms have not been fully understood. Here, we identified a SARS-CoV-2-driven membrane structure named the 3a dense body (3DB). 3DBs are unusual electron-dense and dynamic structures driven by the accessory protein ORF3a via remodeling a specific subset of the trans-Golgi network (TGN) and early endosomal membrane. 3DB formation is conserved in related bat and pangolin coronaviruses but was lost during the evolution to SARS-CoV. During SARS-CoV-2 infection, 3DB recruits the viral structural proteins spike (S) and membrane (M) and undergoes dynamic fusion/fission to maintain the optimal unprocessed-to-processed ratio of S on assembled virions. Disruption of 3DB formation resulted in virions assembled with an abnormal S processing rate, leading to a dramatic reduction in viral entry efficiency. Our study uncovers the crucial role of 3DB in maintaining maximal SARS-CoV-2 infectivity and highlights its potential as a target for COVID-19 prophylactics and therapeutics.
© 2025. The Author(s).
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COVID-19
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Immunology and Microbiology
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STING agonist-based ER-targeting molecules boost antigen cross-presentation.
In Nature on 1 May 2025 by Wang, X., Huang, Z., et al.
CD8+ T cell immune responses are critical for combating infectious diseases and tumours1-3. Antigen cross-presentation, primarily occurring at the endoplasmic reticulum (ER) of dendritic cells, is essential for protein-based vaccines to induce CD8+ T cell responses4. Current efforts have focused on antigen delivery at the tissue and cellular levels, whereas subcellular delivery has been limited to facilitating antigen escape from lysosomes into the cytosol. In the absence of a small-sized high-affinity ER-targeting molecule, the importance of the 'last mile' from the cytosol to the ER remains elusive. Here we developed stimulator of interferon genes (STING) agonist-based ER-targeting molecules (SABER), which effectively deliver antigens to the ER and cluster key machinery in cross-presentation to form microreactors by folding the ER membrane. Conjugation of SABER to various antigens substantially enhances the induction of CD8+ T cell immune responses to tumour neoantigens and conserved viral epitopes, far exceeding that achieved by mixtures of antigens with STING agonists or conventional adjuvants. SABER also retains a potent adjuvant effect, effectively enhancing the ability of a SARS-CoV-2 subunit vaccine to induce broadly neutralizing antibodies. This study provides a high-affinity ER-targeting delivery system and vaccine adjuvant, demonstrating that precise subcellular delivery targeting the last mile of cross-presentation can lead to a qualitative leap.
© 2025. The Author(s).
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Immunology and Microbiology
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In Elife on 16 August 2024 by He, Q., Zhao, M. M., et al.
Fig.6.A
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Immunofluorescent staining of organelle markers representing the endoplasmic reticulum (ER), Golgi apparatus and lysosome.(a) Expression of calreticulin (endoplasmic reticulum marker protein) in WT Huh7 cells. (b) Expression of GM130 (Golgi apparatus marker protein) in WT Huh7 cells. (c) Expressi...
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Immunofluorescent staining of organelle markers representing the endoplasmic reticulum (ER), Golgi apparatus and lysosome.(a) Expression of calreticulin (endoplasmic reticulum marker protein) in WT Huh7 cells. (b) Expression of GM130 (Golgi apparatus marker protein) in WT Huh7 cells. (c) Expression of Lamp1 (lysosome marker protein) in WT Huh7 cells. (d) Mouse immunoglobulin G (IgG) was used as a negative control to show the specificity of the primary antibody binding to GM130 and Lamp1 antigen. (e) Rabbit IgG was used as a negative control to show the specificity of the primary antibody binding to calreticulin antigen.
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Collected and cropped from Elife by CiteAb, provided under a CC-BY license
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In Life Sci Alliance on 1 August 2024 by Veronese, M., Kallabis, S., et al.
Fig.6.E
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ICC-IF
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Homo sapiens (Human)
Double knock-out (DKO) cells show defects along the secretory pathway.(A) Immunofluorescence of the ER using an anti-RTN4 antibody in cells of different genotypes. (B) For quantification of (A), ER tubules were classified as normal, intermediate, and collapsed. N = 3 independent experiments (at l...
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Double knock-out (DKO) cells show defects along the secretory pathway.(A) Immunofluorescence of the ER using an anti-RTN4 antibody in cells of different genotypes. (B) For quantification of (A), ER tubules were classified as normal, intermediate, and collapsed. N = 3 independent experiments (at least 90 cells were analysed per genotype). SD is shown. P-value refers to the collapsed phenotype. (C) RUSH assay using the vesicular stomatitis virus G–GFP construct. Cells were fixed at indicated time points and stained for GM130 to label the Golgi apparatus. (D) Quantification of the percentage of GFP colocalizing with GM130 at each time point. The ratio between the GFP signal inside the GM130-positive area and the total GFP signal is shown. N = 4 biological replicates (at least 110 cells were analysed per genotype for each time point). (E) Immunofluorescence of the Golgi apparatus using an anti-GM130 antibody. (F) Quantification of the experiment as in (E). N = 3 biological replicates (at least 300 cells were analysed per genotype). Graphs show individual data points, mean, and SD. The statistical tests employed are one-way ANOVA with post hoc Tukey’s multiple comparison test.
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Collected and cropped from Life Sci Alliance by CiteAb, provided under a CC-BY license
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In Life Sci Alliance on 1 August 2024 by Veronese, M., Kallabis, S., et al.
Fig.6.C
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ICC-IF
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Homo sapiens (Human)
Double knock-out (DKO) cells show defects along the secretory pathway.(A) Immunofluorescence of the ER using an anti-RTN4 antibody in cells of different genotypes. (B) For quantification of (A), ER tubules were classified as normal, intermediate, and collapsed. N = 3 independent experiments (at l...
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Double knock-out (DKO) cells show defects along the secretory pathway.(A) Immunofluorescence of the ER using an anti-RTN4 antibody in cells of different genotypes. (B) For quantification of (A), ER tubules were classified as normal, intermediate, and collapsed. N = 3 independent experiments (at least 90 cells were analysed per genotype). SD is shown. P-value refers to the collapsed phenotype. (C) RUSH assay using the vesicular stomatitis virus G–GFP construct. Cells were fixed at indicated time points and stained for GM130 to label the Golgi apparatus. (D) Quantification of the percentage of GFP colocalizing with GM130 at each time point. The ratio between the GFP signal inside the GM130-positive area and the total GFP signal is shown. N = 4 biological replicates (at least 110 cells were analysed per genotype for each time point). (E) Immunofluorescence of the Golgi apparatus using an anti-GM130 antibody. (F) Quantification of the experiment as in (E). N = 3 biological replicates (at least 300 cells were analysed per genotype). Graphs show individual data points, mean, and SD. The statistical tests employed are one-way ANOVA with post hoc Tukey’s multiple comparison test.
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Collected and cropped from Life Sci Alliance by CiteAb, provided under a CC-BY license
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In Nat Commun on 27 February 2024 by Tu, Y., Yang, Q., et al.
Fig.3.G
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ICC-IF
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TBC1D23 preferentially regulates the phosphorylation of Golgi-localized proteins, and TBC1D23 loss prevents Golgi disassembly.a Volcano plot of quantitative analysis of phosphopeptides identified by MS. Blue or red: peptides with fold-change (TBC1D23 KO:WT) < 0.67 (p < 0.01, t-test); green or pur...
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TBC1D23 preferentially regulates the phosphorylation of Golgi-localized proteins, and TBC1D23 loss prevents Golgi disassembly.a Volcano plot of quantitative analysis of phosphopeptides identified by MS. Blue or red: peptides with fold-change (TBC1D23 KO:WT) < 0.67 (p < 0.01, t-test); green or purple: peptides with fold-change (TBC1D23 KO:WT) > 1.5 (p < 0.01, t-test); red or purple: golgi-localized proteins; gray: no significant difference. b Combined analysis of phosphosites identified from AMPK DKO (AMPKα1/α2 double knockout) cells and TBC1D23 KO cells. The proportion of Golgi-localized proteins was determined. c Top 10 phosphosites with higher levels of phosphorylation in WT cells than TBC1D23 KO cells, ranked by p value. Golgi-localized proteins were shown in red color. d, e Constructs encoding Flag-tagged ARF1 WT (d) or S147A mutant (e) were transfected into WT or TBC1D23 KO HEK293T cells. 24 h later, cells glucose starved for 2 h were collected and subjected to immunoprecipitation using Flag beads and immunoblotting. The graph showed ratios of bound pST to Flag quantified by densitometry using Image J software. f WT and TBC1D23 KO HEK293T were glucose starved for the indicated time, and cell lysates were subjected to immunoblotting. The graph shows the levels of pGBF1 quantified by densitometry using Image J software and normalized to GBF1. g, h WT and TBC1D23 KO HepG2 cells were treated with or without 10 mM metformin for 4 h. The cells were stained with an anti-GM130 (Golgi marker) antibody. Representative confocal images (g) and quantitation of Golgi elements in cells as treated in (g) (h, left panel). Scale bar, 10 μm. The ratio between the total area occupied by GM130 and DAPI was also used to indicate Golgi disassembly (h, right pannel). Each dot in the figure represents the ratio GM130 area/nucleus area from one cell. Similar results were obtained in three independent experiments (d–h). Results are presented as mean ± SD (d–h). P values were determined by unpaired two-tailed t test (a, c, d, e), or by two-way ANOVA, followed by Sidak’s test (f), or by Scheirer-Ray-Hare Test (h). Source data are provided as a Source data file.
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Collected and cropped from Nat Commun by CiteAb, provided under a CC-BY license
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In PLoS Pathog on 1 October 2023 by Ishida, K., Yagi, H., et al.
Fig.4.G
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ICC-IF
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N-glycosylation-deficient E protein inhibits the ER-Golgi transition of SVPs.(A) Primary structure of a prME expression construct with GFP11 insertion (S275-G11 (E)). The GFP11 tag sequence (green) and GS linker sequence (black) are indicated. (B) Confirmation of SVP formation of GFP11-inserted p...
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N-glycosylation-deficient E protein inhibits the ER-Golgi transition of SVPs.(A) Primary structure of a prME expression construct with GFP11 insertion (S275-G11 (E)). The GFP11 tag sequence (green) and GS linker sequence (black) are indicated. (B) Confirmation of SVP formation of GFP11-inserted prME protein. Cell lysates from the indicated construct-expressing 293T cells were fractionated using 10–40% sucrose density gradient ultracentrifugation and then E protein in each fraction was detected using immunoblotting with anti-E protein antibody. (C) GFP complementation in cells expressing prME (S275-G11 (E)) and GFP1-10. HeLa cells expressing GFP1-10 were transfected with prME WT (left panel) or prME (S275-G11 (E)) (right panel). GFP fluorescence of cells was analyzed using flow cytometry at 24 hours post-transfection. Black and green line in the panels indicates empty and prME vector transfected cell, respectively. (D) Subcellular localization of complemented GFP in cells expressing prME (S275-G11 (E)) and GFP1-10. HeLa cells expressing GFP1-10 were transfected with prME (S275-G11 (E)). At 24 hours post-transfection., cells were fixed and stained with anti-E antibody (red) and Hoechst (Cyan). GFP fluorescence (Green) was observed. Scale bar: 10 μm. (E) Outline of the strategy for the visualization of synchronized transport of SVPs by the RUSH system. In the absence of biotin, ss-Streptavidin-Binding Peptide (SBP) -GFP1-10 reporter with prME (S275-G11 (E)) is retained in the ER by the streptavidin-KDEL hook. After biotin addition, the reconstituted GFP-labeled SVPs leave the ER and translocate to the Golgi apparatus. (F) Timelapse imaging analysis of HeLa cells expressing GFP-labeled SVPs under the RUSH system. Images of GFP fluorescence are shown, and elapsed times after addition of biotin are indicated at the top of each panel. Scale bar: 10 μm. (G) Golgi transport of GFP-labeled SVPs. HeLa cells expressing RUSH-GFP-SVPs WT (top panels) or N154A (bottom panels) were fixed and stained with anti-GM130 (red) at 0 min (left panels) or 30 min (right panels) after biotin addition, and GFP fluorescence (green) was observed. Scale bar: 10 μm. (H) Colocalization of GFP and GM130 signals (G) was determined by calculating Pearson’s correlation coefficient. *p < 0.0001; NS, not significant. Error bars represent standard deviation. Means and SDs from three independent experiments are presented.
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Collected and cropped from PLoS Pathog by CiteAb, provided under a CC-BY license
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In Int J Biol Sci on 16 June 2023 by Ock, J., Wu, J., et al.
Fig.4.B
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WB
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Hebp1 is delivered by MCP-EVs and contributes to nerve regeneration. (A) Representative transmission electron microscopy (TEM) phase images for detecting isolated MCP-EVs. Scale bar = 100 nm. (B) Representative Western blot for Hebp1, EV positive markers (CD63, CD9 and CD81) or EV negative marke...
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Hebp1 is delivered by MCP-EVs and contributes to nerve regeneration. (A) Representative transmission electron microscopy (TEM) phase images for detecting isolated MCP-EVs. Scale bar = 100 nm. (B) Representative Western blot for Hebp1, EV positive markers (CD63, CD9 and CD81) or EV negative marker (GM130) in MCPs and MCP-EVs. (C, D) Expression of Hebp1 in MCP-EVs infected with lentivirus expressing shHebp1 or shCon at three doses (1 × 103, 5 × 104, and 1 × 104 TU/ml culture medium) for at least 3 days. (E, F) Densitometric quantification of Hebp1 protein bands using an image analyzer. Results are presented as means ± SEM (n = 4). (G, H) Immunofluorescence staining for NF in mouse MPG tissues exposed to LPS (2.5 μg/ml) and other indicated conditions for 5 days (G). Lengths of NF-positive neurites in MPG tissues, quantified using an image analyzer (H). Results are presented as means ± SEM (n = 4). Scale bar, 100 µm. (I, J) Migration assays of mouse Schwann cells exposed to LPS (2.5 μg/ml) and other indicated conditions for 24 hours (I). Number of migrated cells in the red dotted rectangle, quantified using an image analyzer (J). Results are presented as means ± SEM (n = 4; **P < 0.01, ***P < 0.001). The relative ratio of the shCon (control) group was defined as 1. MCPs, mouse cavernous pericytes; EVs, extracellular vesicles; LPS, lipopolysaccharide; MPG, major pelvic ganglion; PBS, phosphate-buffered saline; ns, not significant.
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Collected and cropped from Int J Biol Sci by CiteAb, provided under a CC-BY license
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In Sci Rep on 4 April 2023 by Kim, J. A., Park, C., et al.
Fig.1.C
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WB
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Homo sapiens (Human)
Characterization of serum-derived small extracellular vesicles (sEV). (A) Representative transmission electron microscopy (TEM) images of sEV isolated by exoEasy kit. Scale bars are 500 nm on the left and middle images (magnification × 60,000) and 200 nm on the right image (magnification × 100,00...
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Characterization of serum-derived small extracellular vesicles (sEV). (A) Representative transmission electron microscopy (TEM) images of sEV isolated by exoEasy kit. Scale bars are 500 nm on the left and middle images (magnification × 60,000) and 200 nm on the right image (magnification × 100,000). Spherical membranous particles with an expected size of sEV are indicated with red arrows. (B) Representative particles size distribution profile of nanoparticle tracking analysis (NTA). The calculated size distribution is depicted as a mean (black line) with standard error (red shaded area) along with descriptive summary of particles size. The mean concentration of particles (± standard error) was 2.73 × 109 (5.71 × 107) particles/ml. Data were analyzed using NanoSight NTA software v3.2 (https://www.malvernpanalytical.com/en/support/product-support/software/nanosight-nta-software-update-v3-2). (C) The expression of CD63 (exosome marker) and transferrin (negative marker) in sEV-enriched eluate and sEV-depleted flow-through was determined by western blotting. The expression of calnexin and GM130 (both negative markers) was also examined in sEV eluate and cell lysate (HEK293T). Full scans of all uncropped images are provided in Supplementary Fig. 1.
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Collected and cropped from Sci Rep by CiteAb, provided under a CC-BY license
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In IBRO Neurosci Rep on 1 December 2022 by Nakamura, A., Ikeda, M., et al.
Fig.2.D
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ICC-IF
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Δe17 isoform localized diffusely in cytoplasm and binds to γTuRC in HEK293T cells. Cells were observed with a laser confocal microscope. (A) Immunostaining with anti-CDK5RAP2 antibody and GM130 antibody (Golgi marker). Endogenous CDK5RAP2 localized intensely at the centrosome (arrow) and weakly o...
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Δe17 isoform localized diffusely in cytoplasm and binds to γTuRC in HEK293T cells. Cells were observed with a laser confocal microscope. (A) Immunostaining with anti-CDK5RAP2 antibody and GM130 antibody (Golgi marker). Endogenous CDK5RAP2 localized intensely at the centrosome (arrow) and weakly on the Golgi apparatus. (B) Anti-FLAG immunostaining of FLAG-tagged full-length CDK5RAP2. The full-length isoform localized intensely at the centrosome (arrow) and weakly on the Golgi apparatus. (C) Localization of FLAG-tagged Δe17 isoform in cells expressing low level of the isoform. FLAG immunostaining showed diffuse localization of the isoform in the cytoplasm as well as weak localization at the centrosome (arrow). (D) Localization of FLAG-tagged Δe17 isoform in cells expressing low level of the isoform. FLAG immunostaining did not show obvious localization of the isoform at the Golgi apparatus (arrow). (E) Localization of FLAG-tagged Δe17 isoform in cells expressing a high level of the isoform. FLAG immunostaining showed diffuse and even localization in the cytoplasm. (F) Interaction of Δe17 isoform with γTuRC. Lysates of cells transfected with FLAG-tagged CDK5RAP2 isoforms were immunoprecipitated with FLAG antibody and were immunoblotted with anti-γ-tubulin antibody and with anti-MZT2 antibody.
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Collected and cropped from IBRO Neurosci Rep by CiteAb, provided under a CC-BY license
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In IBRO Neurosci Rep on 1 December 2022 by Nakamura, A., Ikeda, M., et al.
Fig.3.C
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ICC-IF
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MT regrowth experiments. (A, B) HEK293T cells were treated with cold-nocodazole, washed out, and incubated to allow MT regrowth. Regrown MTs were detected by α-tubulin antibody. Pericentrin antibody stained the centrosome and GM130 antibody stained the Golgi apparatus. Cells were observed with a ...
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MT regrowth experiments. (A, B) HEK293T cells were treated with cold-nocodazole, washed out, and incubated to allow MT regrowth. Regrown MTs were detected by α-tubulin antibody. Pericentrin antibody stained the centrosome and GM130 antibody stained the Golgi apparatus. Cells were observed with a laser confocal microscope. MT regrowth was observed at the centrosome (arrow in A) and the Golgi apparatus (B). (C) Neuro2A cells were treated as in (B) to show similar regrowth pattern of MTs as HEK293T cells. (D, E) Triple immunostaining of cells for FLAG, regrown MTs (α-tubulin), and the centrosome (pericentrin) or the Golgi apparatus (GM130). Cells expressing FLAG-tagged Δe17 isoform exhibited evenly distributing regrown MTs throughout the cytoplasm. Some of them were colocalized with the centrosome (arrow in D) or the Golgi apparatus (arrows in E), but others were not associated with the centrosome nor with the Golgi apparatus. (F) Neuro2A cells transfected with GFP-tagged Δe17 isoform. A similar distribution of regrown MTs to (D) was observed. Arrow indicates the centrosome. (G) The number of regrown MTs was counted in control cells and FLAG-tagged Δe17 isoform-transfected HEK293T cells. The graph is shown with the tukey method of box and whisker plot. ***p < 0.001.
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Collected and cropped from IBRO Neurosci Rep by CiteAb, provided under a CC-BY license
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In J Biol Chem on 1 August 2022 by Januário, Y. C., Eden, J., et al.
Fig.3.G
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ICC-IF
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Residue Y682 of the APP tail is essential for proper localization of APP.A, schematic representation of APP fused with either GFP or mCherry at the C terminus and its processing products. APP fragments after processing include C99, C83, and AICD-γ, all fused with GFP or mCherry. The recognition s...
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Residue Y682 of the APP tail is essential for proper localization of APP.A, schematic representation of APP fused with either GFP or mCherry at the C terminus and its processing products. APP fragments after processing include C99, C83, and AICD-γ, all fused with GFP or mCherry. The recognition site of 6E10 antibody is indicated in full-length APP and C99. APP was expressed with a F615P substitution to reduce α-secretase cleavage. DAPT was used to inhibit γ-secretase activity and facilitate C99 fragment visualization. B, confocal microscopy images of H4 neuroglioma cells co-transfected with APP WT-mCherry and APP Y682A-GFP and immunolabeled with anti-TGN46. Green arrows indicate puncta exclusively of APP/CTFs Y682A-GFP. Red arrows highlight puncta with only APP/CTFs WT-mCherry. C–F, APP/CTFs WT-GFP and APP/CTFs Y682A-GFP co-localization with markers measured using Pearson’s coefficient. Values represent mean ± SEM from at least eight different cells. G, rat cortical neurons co-transfected with APP WT-mCherry and APP Y682A-GFP and immunolabeled with anti-GM130. H, APP/CTFs WT-mCherry and APP/CTFs Y682A-GFP co-localization compared in the same transfected neuron using Pearson’s coefficient. Merge channel is the combination of APP WT-mCherry, APP Y682A-GFP, and GM130 channels. Main panels scale bar represents 10 μm; insets (2×) scale bar represents 2.5 μm. ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001. Statistical significance was calculated by two-tailed Student's t test in C–F and H. APP, amyloid precursor protein; TGN, trans-Golgi network.
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Collected and cropped from J Biol Chem by CiteAb, provided under a CC-BY license
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In J Biol Chem on 1 August 2022 by Januário, Y. C., Eden, J., et al.
Fig.6.D
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ICC-IF
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Expression of dominant-negative μ1A W408S increases APP/CTF association with Golgi markers in H4 cells and neurons.A and B, confocal microscopy images of H4 cells transfected with μ1A WT-HA or μ1A W408S-HA, immunolabeled with anti-C99 (to stain endogenous APP and C99), anti-HA, and anti-TGN46 ant...
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Expression of dominant-negative μ1A W408S increases APP/CTF association with Golgi markers in H4 cells and neurons.A and B, confocal microscopy images of H4 cells transfected with μ1A WT-HA or μ1A W408S-HA, immunolabeled with anti-C99 (to stain endogenous APP and C99), anti-HA, and anti-TGN46 antibodies. Merge channel is a combination of APP/C99 and TGN46 channels. C–E, rat cortical neurons transfected with APP-mCherry in combination with GFP (C), μ1A WT-GFP (D), or μ1A W408S-GFP (E). Cells were immunolabeled with anti-GM130. F, endogenous APP/C99 and TGN46 co-localization in H4 cells expressing μ1A WT-HA or μ1A W408S-HA was measured using Fiji software with Pearson’s coefficient. Values represent mean ± SEM from at least 15 different cells. G, APP/CTFs-mCherry and GM130 co-localization in neurons expressing μ1A W408S-GFP, μ1A WT-GFP, or GFP alone. Values represent mean ± SEM from at least eight different cells. Main panels scale bar represent 10 μm; insets (2×) scale bar represents 2.5 μm. ∗p ≤ 0.05; ∗∗p ≤ 0.01; and ∗∗∗p ≤ 0.001. Statistical significance was calculated by two-tailed Student’s t test in F and one-way ANOVA followed by Tukey post-test in G. APP, amyloid precursor protein; CTF, carboxyl-terminal fragment; HA, hemagglutinin; TGN, trans-Golgi network.
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Collected and cropped from J Biol Chem by CiteAb, provided under a CC-BY license
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In Sci Rep on 11 June 2022 by Zorn, M., Kühnisch, J., et al.
Fig.2.A
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ICC-IF
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Homo sapiens (Human)
Golgi enrichment is affected by VPS13B missense variants. (A–D) Golgi fragmentation is rescued by wild type VPS13B overexpression. HeLa cells were transfected with either control (scrambled siRNA) (A,B) or a VPS13B-specific RNAi (siRNA #25721, ambion) (C,D). 48 h post RNAi-transfection HeLa cells...
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Golgi enrichment is affected by VPS13B missense variants. (A–D) Golgi fragmentation is rescued by wild type VPS13B overexpression. HeLa cells were transfected with either control (scrambled siRNA) (A,B) or a VPS13B-specific RNAi (siRNA #25721, ambion) (C,D). 48 h post RNAi-transfection HeLa cells were processed for overexpression and either nontransfected (A,C) or transfected with the pcDNA3.1_VPS13B-wt construct (B,D). 24 h later, all cells were processed for immunostaining of VPS13B (green) and GM130 (red). Imaging occurred with confocal microscopy. Nuclei were stained using DAPI, scale bars 10 µm. (E,F) Analysis of subcellular localization of cloned VPS13B missense variants. ImageJ analysis was used to evaluate Golgi localization of VPS13B missense variants. Two ROI (yellow) were defined for each cell: “whole cell” ROI was drawn at the outline of the cell to detect total immuofluorescence levels; “Golgi region” was defined by the counterstaining of GM130 (red). Fluorescence intensity of VPS13B (green) in each ROI was measured (E). Percentage of VPS13B enrichment at the Golgi was finally calculated for each missense variant using GraphPad prism. We repeated each condition at least 3 times and analyzed each time 3 recordings (with at least 1 transfected cell). Please refer to Supplementary table T3 for statistical information (F).
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Collected and cropped from Sci Rep by CiteAb, provided under a CC-BY license
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In Sci Rep on 11 June 2022 by Zorn, M., Kühnisch, J., et al.
Fig.2.D
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ICC-IF
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Homo sapiens (Human)
Golgi enrichment is affected by VPS13B missense variants. (A–D) Golgi fragmentation is rescued by wild type VPS13B overexpression. HeLa cells were transfected with either control (scrambled siRNA) (A,B) or a VPS13B-specific RNAi (siRNA #25721, ambion) (C,D). 48 h post RNAi-transfection HeLa cells...
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Golgi enrichment is affected by VPS13B missense variants. (A–D) Golgi fragmentation is rescued by wild type VPS13B overexpression. HeLa cells were transfected with either control (scrambled siRNA) (A,B) or a VPS13B-specific RNAi (siRNA #25721, ambion) (C,D). 48 h post RNAi-transfection HeLa cells were processed for overexpression and either nontransfected (A,C) or transfected with the pcDNA3.1_VPS13B-wt construct (B,D). 24 h later, all cells were processed for immunostaining of VPS13B (green) and GM130 (red). Imaging occurred with confocal microscopy. Nuclei were stained using DAPI, scale bars 10 µm. (E,F) Analysis of subcellular localization of cloned VPS13B missense variants. ImageJ analysis was used to evaluate Golgi localization of VPS13B missense variants. Two ROI (yellow) were defined for each cell: “whole cell” ROI was drawn at the outline of the cell to detect total immuofluorescence levels; “Golgi region” was defined by the counterstaining of GM130 (red). Fluorescence intensity of VPS13B (green) in each ROI was measured (E). Percentage of VPS13B enrichment at the Golgi was finally calculated for each missense variant using GraphPad prism. We repeated each condition at least 3 times and analyzed each time 3 recordings (with at least 1 transfected cell). Please refer to Supplementary table T3 for statistical information (F).
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Collected and cropped from Sci Rep by CiteAb, provided under a CC-BY license
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In Nat Commun on 1 June 2022 by Byron, A., Griffith, B. G. C., et al.
Fig.2.B
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WB
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Mus musculus (House mouse)
Characterisation of a nucleo-adhesome.a Methodological workflow for subcellular fractionation and enrichment of nuclei. DOC, sodium deoxycholate; TX-100, Triton X-100. b Effective subcellular fractionation of SCC cells. Markers for given subcellular locations are indicated. Immunoblots are repres...
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Characterisation of a nucleo-adhesome.a Methodological workflow for subcellular fractionation and enrichment of nuclei. DOC, sodium deoxycholate; TX-100, Triton X-100. b Effective subcellular fractionation of SCC cells. Markers for given subcellular locations are indicated. Immunoblots are representative of five independent experiments. c Workflow for mass spectrometric characterisation of cytoplasmic, perinuclear and nuclear subproteomes of SCC cells. d Numbers of proteins identified in each subcellular fraction. Black bar, median; light grey box, range; circle, replicate data point (n = 5 independent biological replicates). Numbers of proteins identified in at least four out of five biological replicate subcellular fractions are represented as bars. Purple dashed lines indicate numbers of proteins also identified in nuclear fractions (regardless of relative abundance). e Principal component analysis of proteins quantified in at least four out of five biological replicate experiments. f Hierarchical cluster analysis of the cytoplasmic, perinuclear and nuclear subproteomes. Relative protein abundance was min-max scaled protein-wise (scaled intensity). Memberships of the consensus adhesome and literature-curated adhesome are indicated (bins, 50 proteins). g t-SNE map of the subcellular proteomes (Supplementary Data 2) annotated with curated subcellular markers and consensus adhesome proteins. h Confocal imaging of SCC cells in the presence or absence of 10 nM leptomycin B (LMB). Nuclei were detected using NucBlue. z-slices passing through the centres of the nuclei (greyscale channels) are shown alongside maximum intensity projections (merged channels). Inverted lookup tables were applied; in merged images, colocalisation of paxillin (magenta) and NucBlue (green) is represented by black regions. Images are representative of three independent experiments. Scale bars, 20 μm. i Quantification of nuclear paxillin signal in nucleus-central z-slices (see h). Black bars, condition mean (thick bar) ± s.d. (thin bars); grey silhouette, probability density. Data from different biological replicates (rep.) are indicated by coloured circles; replicate means are indicated by large circles. Statistical analysis, two-sided Welch’s t-test of replicate means (n = 52 and 57 cells for control and LMB treatment, respectively, from n = 3 independent biological replicates). Source data are provided as a Source Data file.
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Collected and cropped from Nat Commun by CiteAb, provided under a CC-BY license
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In Cell Death Dis on 20 December 2021 by Khan, S., Sbeity, M., et al.
Fig.1.C
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ICC-IF
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CRISPR/Cas9 knockdown of TMEM165.A Alignment of TMEM165 targeted sequence from wild-type and mutant mouse ATDC5 cells. B Detection of TMEM165 in cell lysates from wild-type and tmem165-knockout mouse ATDC5 cells (mutant 1, 2, 3 and 4) using anti-TMEM165 specific antibodies. β-actin was used as lo...
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CRISPR/Cas9 knockdown of TMEM165.A Alignment of TMEM165 targeted sequence from wild-type and mutant mouse ATDC5 cells. B Detection of TMEM165 in cell lysates from wild-type and tmem165-knockout mouse ATDC5 cells (mutant 1, 2, 3 and 4) using anti-TMEM165 specific antibodies. β-actin was used as loading control (n = 3). C Immunofluorescence analysis of the expression of TMEM165 in mouse ATDC5 control and tmem165-knockout cells using antibodies against TMEM165 (green). GM130 (red) was used as a Golgi marker. The nucleus was stained with DAPI (blue) (n = 3). Digital images were captured with an inverted microscope, Leica DMI3000. Representative images from three independent experiments are shown. Scale bar: 50 μm.
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Collected and cropped from Cell Death Dis by CiteAb, provided under a CC-BY license
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In Nat Commun on 19 March 2021 by Zhang, Y., Varela, L., et al.
Fig.9.B
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The Kv3.3 G592R mutation promotes release of exosomes into the extracellular space.a Representative transmission electron microscopy photomicrographs of low density fraction (LDF) and intermediate-density fraction (IDF) extracellular vesicles (EVs) isolated from the cerebellum of a wild type mous...
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The Kv3.3 G592R mutation promotes release of exosomes into the extracellular space.a Representative transmission electron microscopy photomicrographs of low density fraction (LDF) and intermediate-density fraction (IDF) extracellular vesicles (EVs) isolated from the cerebellum of a wild type mouse. Scale bar: 500 nm. Images are representative of ten images taken of each of the sets analyzed. b Representative Western blot analyses of LDF EVs (L) and IDF EVs (I) compared to the cerebellar homogenate (BH). c Nanotrack analysis (NTA) of the hydrodynamic diameter of the EVs found in LDFs and IDFs. The curve is normalized to the mode of each distribution (five independent isolations. d Quantification of particles in LDFs and IDFs by NTA that are found within the size bins shown in the graph. Data are plotted as percentage of total number of EVs. Five independent isolations. e Representative Western blot analyses of EVs isolated from cerebella of wild type and G592R Kv3.3 mutant mice. LDFs (L) and IDFs (I) were isolated from cerebella of two mice of the same genotype and these were combined and tested for the markers shown. Two independent experiments are shown in the blot. f–h quantification of data in e. The densitometric quantification was performed on three independent experiments, representative of six mice per genotype. BCA protein assay (i) and total particle count as estimated by NTA (j) of LDFs and IDFs in wild type and G592R Kv3.3 mice. Values were normalized to the cerebellum wet weight (five independent isolations for wild type and three independent isolations for mutant mice, corresponding to ten and six mice, respectively. All data are shown as a mean ± SEM. Statistical test used in all figures: Two-Way ANOVA with Sidak’s multiple comparisons test). Source data and uncropped Western blots are provided as a Source Data file.
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Collected and cropped from Nat Commun by CiteAb, provided under a CC-BY license
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In Front Immunol on 24 November 2020 by Yu, J., He, X., et al.
Fig.6.A
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Protein level and subcellular content analysis of v-SNARE and Rab proteins in mice crypts. (A) Representative image of 5~50% Optiprep gradient of ep/+ and ep mice small intestine crypts. VAMP7 mainly appears at the 11th and 12th fractions labeled by lysozyme and endosomal component TfR and a seco...
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Protein level and subcellular content analysis of v-SNARE and Rab proteins in mice crypts. (A) Representative image of 5~50% Optiprep gradient of ep/+ and ep mice small intestine crypts. VAMP7 mainly appears at the 11th and 12th fractions labeled by lysozyme and endosomal component TfR and a second peak appears at the first two fractions labeled by GM130 and TGN38, while it increases in the 4th fraction in ep crypts. Rab38 mainly appears at the first two fractions of both genotypes while its concentration increases in the middle fragments in ep crypts. Rab32 has similar distributional pattern as Rab38 but more widely distributed in 3rd~6th fractions, but less in the 11th fraction labeled by TfR in ep crypts. (B) Statistical analysis of Rab38 distribution in (A). In ep crypt, the increase of Rab38 in the middle density fragments is significant (the 3rd~5th fractions, ep/+ crypts 9%, ep crypts 16%, *p < 0.05). (C) Statistical analysis of Rab32 distribution in (D). Rab32 in ep crypts significantly decreases at the low density fragment (the 1st fraction: ep/+ crypts 25%, ep crypts 16%, *p < 0.05) and at the 9th fraction (ctrl 4%, ep 2%, *p < 0.05). (D) Statistical analysis of VAMP7 distribution in (A). VAMP7 in ep crypts significantly increases in TGN38 and GM130 enriched fraction (the 2nd fraction, ctrl crypts 17%, ep crypts 28%, *p < 0.05) and the 4th fraction (ep/+ crypts 2%, ep crypts 6%, **p < 0.01), and decreases in the RE enriched 11th fraction (ep/+ crypts 18%, ep crypts 5%, *p < 0.05). 4 mice of each genotype were analyzed, values are means ± s.e.m. of three replicates of immunoblotting data.
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Collected and cropped from Front Immunol by CiteAb, provided under a CC-BY license
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In Front Immunol on 24 November 2020 by Yu, J., He, X., et al.
Fig.7.A
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IHC-IF
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Altered subcellular location of VAMP7 in ep Paneth cells. (A) Representative immunofluorescence images of ep/+ (Ctrl) and ep ileum section stained with anti-VAMP7 (green) and anti-GM130 (purple). UEA1 (red) indicates LDCVs. The arrow heads indicate the co-localization of VAMP7 and GM130, the open...
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Altered subcellular location of VAMP7 in ep Paneth cells. (A) Representative immunofluorescence images of ep/+ (Ctrl) and ep ileum section stained with anti-VAMP7 (green) and anti-GM130 (purple). UEA1 (red) indicates LDCVs. The arrow heads indicate the co-localization of VAMP7 and GM130, the open triangles indicate the co-localization of VAMP7 and UEA-1 and the white arrows indicate the co-localization of all those three makers. (B) Boxed areas are shown at higher magnification. Bar = 5 μm.
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Collected and cropped from Front Immunol by CiteAb, provided under a CC-BY license
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In Front Immunol on 24 November 2020 by Yu, J., He, X., et al.
Fig.7.B
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IHC-IF
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Altered subcellular location of VAMP7 in ep Paneth cells. (A) Representative immunofluorescence images of ep/+ (Ctrl) and ep ileum section stained with anti-VAMP7 (green) and anti-GM130 (purple). UEA1 (red) indicates LDCVs. The arrow heads indicate the co-localization of VAMP7 and GM130, the open...
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Altered subcellular location of VAMP7 in ep Paneth cells. (A) Representative immunofluorescence images of ep/+ (Ctrl) and ep ileum section stained with anti-VAMP7 (green) and anti-GM130 (purple). UEA1 (red) indicates LDCVs. The arrow heads indicate the co-localization of VAMP7 and GM130, the open triangles indicate the co-localization of VAMP7 and UEA-1 and the white arrows indicate the co-localization of all those three makers. (B) Boxed areas are shown at higher magnification. Bar = 5 μm.
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Collected and cropped from Front Immunol by CiteAb, provided under a CC-BY license
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In Medicines (Basel) on 6 May 2020 by Takeuchi, Y., Tanaka, M., et al.
Fig.9.D
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ICC-IF
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The 292CA mutant proteins interact with actin and treatment with ibuprofen decreases this interaction. (A) The total amounts (100%) or 30% of all immunoprecipitates from human 293T cell lysates expressing the control FLAG-GFP or the 292CA-specific de novo peptide with FLAG-GFP tag (right upper le...
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The 292CA mutant proteins interact with actin and treatment with ibuprofen decreases this interaction. (A) The total amounts (100%) or 30% of all immunoprecipitates from human 293T cell lysates expressing the control FLAG-GFP or the 292CA-specific de novo peptide with FLAG-GFP tag (right upper letters) were subjected to a denatured polyacrylamide gel and the gel was silver-stained (left image). The resultant peptides digested from an approximately 50-kDa protein specific to 292CA were analyzed with an Ettan MALDI-TOF Pro mass spectroscope and identified as human actin (β type actin, right bottom letters with red color [mass spectroscopically-identified peptides]). The single and double white circles indicate proteins corresponding to actin position and bait, respectively. (B) N1E-115 cells were transfected with the plasmid encoding the wild type construct or the 292CA or Q39X mutant of GFP-tagged AIMP1 and lysed. The lysates were immunoprecipitated (IP) with an anti-GFP antibody and immunoblotted (IB) with an anti-actin antibody (upper panel). The lysates were also immunoblotted with an antibody against GFP or actin (middle or bottom panel). (C) N1E-115 cells transfected with the respective GFP-tagged AIMP1 constructs (green) were immunostained with an anti-actin antibody (red). Merged images are also shown. The arrows indicate actin fibers in the cell periphery, which are seen only in the case of transfection with the wild type. The scale bars indicate 20 μm. (D) Lysates of transfected N1E-115 cells in culture with or without ibuprofen were immunoprecipitated with an anti-SLC38A9 antibody (for the lysosome) and immunoblotted with an antibody against GFP (for each of the AIMP1 constructs), actin, SLC39A9, LAMP1, KDEL antigen, or GM130. The immunoblots for GFP-tagged AIMP1 proteins (the wild type or each of the mutants), KDEL antigen, or GM130 for total lysates are shown.
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Collected and cropped from Medicines (Basel) by CiteAb, provided under a CC-BY license
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In EMBO Rep on 6 May 2020 by Shizukuishi, S., Ogawa, M., et al.
Fig.3.E
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ICC-IF
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CbpC could bind to p62 and act as a decoy for autophagic degradation of Atg14 to suppress autophagic degradationALysates from MEFs stably expressing HA‐Atg14 infected with S. pneumoniae TIGR4 WT or ΔcbpC for 1, 2, or 3 h in the presence or absence of cycloheximide were subjected to SDS–PAGE and a...
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CbpC could bind to p62 and act as a decoy for autophagic degradation of Atg14 to suppress autophagic degradationALysates from MEFs stably expressing HA‐Atg14 infected with S. pneumoniae TIGR4 WT or ΔcbpC for 1, 2, or 3 h in the presence or absence of cycloheximide were subjected to SDS–PAGE and analyzed by immunoblotting with the indicated antibodies.BMEFs/HA‐Atg14 cells were infected with S. pneumoniae TIGR4 WT or ΔcbpC for 1, 2, or 3 h in the presence or absence of Bafilomycin A1 (BafA1). The lysates were subjected to SDS–PAGE and analyzed by immunoblotting with the indicated antibodies.C–F(C, E) A549 cells were infected with S. pneumoniae R6 WT or ΔcbpC for 2 h and fixed and stained with DAPI and an anti‐Atg14 or anti‐GM130 antibody. Representative epifluorescence images are shown. Scale bars, 10 μm. (D, F) The percentages of perinuclear‐localizing Atg14 containing cells in (C) or intact Golgi apparatus‐containing cells in (E) were quantified.GStreptococcus pneumoniae (Sp) invasion‐permissive A549 cells (marked with GFP) and Sp invasion‐non‐permissive (pIgR knocked down) A549 cells were co‐cultured at a 1:3 ratio. Atg14‐disappearance experiments were conducted, and representative epifluorescence images are shown. Scale bars, 10 μm. The lines show each cell shape, and the arrows show invasion‐permissive A549 cells marked with GFP.HThe percentages of perinuclear‐localizing Atg14 containing cells in (G) were quantified.ILysates from 293T cells transiently expressing GFP‐CbpC or GFP, and HA‐Atg14 were subjected to SDS–PAGE and analyzed by immunoblotting using antibodies against HA or GFP.JLysates from 293T cells transiently expressing GFP‐CbpCT4 or GFP were immunoprecipitated using GST‐GFP‐Nanobody. Additionally, beads were mixed with lysates from 293T cells transiently expressing p62‐3Myc and HA‐Atg14, and bound proteins were analyzed by immunoblotting.KA549 cells treated with the indicated siRNAs were infected with S. pneumoniae R6 WT or ΔcbpF for the indicated durations. The cells were fixed and stained with DAPI and an anti‐Atg14 antibody, and percentages of perinuclear‐localizing Atg14 containing cells were quantified.LLysates from MEFs stably expressing GFP‐CbpCT4 or GFP in the presence or absence of rapamycin or chloroquine were subjected to SDS–PAGE and immunoblotted using antibodies against LC3 or p62.MQuantification of NanoBRET signals in 293A cells transiently expressing HaloTag‐Stx17 and Nanoluc‐Atg14 in the presence or absence of GFP‐CbpC or GFP.Data information: In (D, F, H, K, M), data represent mean ± SEM of 3 biological replicates. Student's t‐test was used to calculate statistical significance. *P < 0.01, N.S., not significant.Source data are available online for this figure.
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Collected and cropped from EMBO Rep by CiteAb, provided under a CC-BY license
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In Nat Commun on 29 April 2020 by Sung, B. H., von Lersner, A., et al.
Fig.2.C
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pHluo_M153R-CD63 labels exosomes.a Averaged trace from nanoparticle tracking analyses of large EVs (red) and small EVs (green) secreted from pHluo_M153R-CD63-expressing HT1080. Shaded area represents s.e.m. from three independent experiments. Small EVs were purified through iodixanol density grad...
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pHluo_M153R-CD63 labels exosomes.a Averaged trace from nanoparticle tracking analyses of large EVs (red) and small EVs (green) secreted from pHluo_M153R-CD63-expressing HT1080. Shaded area represents s.e.m. from three independent experiments. Small EVs were purified through iodixanol density gradient. b Representative immunogold negative stain electron micrograph of small EVs iodixanol density-gradient-purified from pHluo_M153R-CD63-expressing HT1080 from two independent experiments. Scale bars, 100 nm. c Western blot analysis of cells and EVs with anti-CD63, anti-GFP, EV markers (Flotillin, HSP70) and Golgi marker (GM130). L total cell lysate, lEV large EV, sEV small EV. d Immunoblotting of Rab27a in pHluo_M153R-CD63-stably expressing HT1080. Sc scrambled control, KD knockdown. e Comparison of small EV (sEV) secretion rate between control and Rab27a-KDs shown as a scatter-plot (mean ± standard error) from three independent experiments. *P = 0.030793 and 0.031077 for Rab27a-KD1 and KD2, respectively, by two-sided paired Student’s t test. f−h Live imaging and analysis of extracellular pHluo_M153R-CD63 for control and Rab27a-KD cells. f Representative images, n = 31 images from three independent experiments for each cell line. R27a, Rab27a. Scale bar, 30 µm. g Threshold area of the extracellular region from (f) shown as a scatter-plot (median and quartile range). ***P < 0.001 by two-sided unpaired Mann−Whitney U test. h Integrated intensity of the extracellular region from (f) shown as a scatter-plot (median and quartile range). ***P < 0.001 by two-sided unpaired Mann−Whitney U test. Source data are provided as a Source Data file.
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Collected and cropped from Nat Commun by CiteAb, provided under a CC-BY license
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In J Clin Med on 23 April 2020 by Santos, M., Damásio, J., et al.
Fig.3.A
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ICC-IF
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Homo sapiens (Human)
MAG-C42R has an altered subcellular localization. (A) The localization of EGFP-tagged MAG clones in HEK293T cells. EGFP was directly visualized. The presence of MAG in the endoplasmic reticulum and Golgi complex was analyzed by probing cells with anti-calnexin and anti-GM130 antibodies, respectiv...
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MAG-C42R has an altered subcellular localization. (A) The localization of EGFP-tagged MAG clones in HEK293T cells. EGFP was directly visualized. The presence of MAG in the endoplasmic reticulum and Golgi complex was analyzed by probing cells with anti-calnexin and anti-GM130 antibodies, respectively, both detected with Alexa Fluor 568-conjugated secondary antibodies (red). DNA was stained with Hoechst 33342 (blue). Photographs were acquired using a Zeiss Axio Imager Z1 microscope. Bars: 10 μm. (B) The co-immunoprecipitation of EGFP-tagged MAG clones with endogenous calnexin and GM130 in HEK293T cells. Cell lysates were immunoprecipitated with anti-GFP antibody. The immunoprecipitates were subjected to immunoblotting with anti-GM130 antibody, then stripped and reprobed with anti-calnexin antibody, followed by stripping and reprobing with anti-EGFP antibody. The same methodology was applied to whole cell lysates. Calnexin and GM130 immunoblotting figures have different exposure times (original blots are presented in Figure S2).
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Collected and cropped from J Clin Med by CiteAb, provided under a CC-BY license
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In J Cell Biol on 2 March 2020 by Larios, J., Mercier, V., et al.
Fig.8.A
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ALIXΔPRR stimulates the release of exosomes containing tetraspanins. (A and B) HeLa GFP-CHMP4B cells transfected for 18 h with ALIXΔPRR (ΔPRR) or GFP as a control and incubated with exosome-free medium for 24 h. The cell medium was collected, and exosomes were isolated by differential centrifugat...
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ALIXΔPRR stimulates the release of exosomes containing tetraspanins. (A and B) HeLa GFP-CHMP4B cells transfected for 18 h with ALIXΔPRR (ΔPRR) or GFP as a control and incubated with exosome-free medium for 24 h. The cell medium was collected, and exosomes were isolated by differential centrifugation. The cell lysate and the exosome samples were analyzed by Western blotting using antibodies against the indicated proteins. The same amount of protein was loaded for each condition (A). The amounts of protein were quantified by densitometry and are expressed relative to free GFP. In B, boxes, mean; error bars, ±SD (n = 3, from three independent experiments); t test; *, P < 0.05, ***, P < 0.001; ns, not significant. (C and D) HeLa GFP-CHMP4B cells were treated with siRNA against ALIX or nontarget siRNAs (siCtrl) and incubated with exosome-free medium for 24 h. Exosomes were isolated as in A and B, and then cell lysates and exosomes (C) were analyzed by Western blotting as in A and B. The relative amount of protein in exosome fractions was quantified by densitometry (D). Boxes, mean; error bars, ±SD (n = 3, from three independent experiments); t test; *, P < 0.05, ***, P < 0.001; ns, not significant. (E and F) The experiment was as in C and D except that siRNAs against CHMP6 and not against ALIX were used. The upper band in the syntenin blot of siCtrl is CHMP6, because the same membrane was reused for incubation with both antibodies. The relative amounts of protein were then quantified by densitometry (F). Boxes, mean; error bars, ±SD (n = 3, from three independent experiments); t test; *, P < 0.05, **, P < 0.01. (G and H) HeLa GFP-CHMP4B cells were transfected with WT CD9 (WT) tagged with V5 or with the V5-CD9/3R mutant (3R). Total cell lysates and exosomes prepared as in A and B were analyzed by Western blotting using antibodies against V5, ALIX, flotillin-1, and syntenin (G). The V5 blot is shown after short and long exposure, so that WT V5-CD9 and V5-CD9/3R can be better compared. The relative amounts of CD9 were quantified by densitometry (H). Boxes, mean; error bars, ±SD (n = 3, from three independent experiments); t test; *, P < 0.05.
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Collected and cropped from J Cell Biol by CiteAb, provided under a CC-BY license
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In Front Mol Neurosci on 11 February 2020 by He, Q., Liu, H., et al.
Fig.6.A
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Overexpression of GM130 ameliorates apoptosis, restores the protein levels of occludin and claudin 5, and reduces Golgi fragmentation during HSV-1 infection. Bend.3 cells were transfected with either a negative control plasmid pcDNA3.1 (NC) or GM130 expression plasmid pcDNA3.1-GM130 (OE). Twelve ...
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Overexpression of GM130 ameliorates apoptosis, restores the protein levels of occludin and claudin 5, and reduces Golgi fragmentation during HSV-1 infection. Bend.3 cells were transfected with either a negative control plasmid pcDNA3.1 (NC) or GM130 expression plasmid pcDNA3.1-GM130 (OE). Twelve hours after transfection, cells were infected with HSV-1 for 36 h and then harvested. (A) GM130 and cleaved-caspase 3 protein levels were determined by immunoblotting. GM130 and cleaved-caspase 3 protein levels in HSV-1-infected cells transfected with pcDNA3.1 or pcDNA3.1-GM130 were normalized to the levels in mock-infected cells transfected with pcDNA3.1 or pcDNA3.1-GM130, respectively, to provide fold changes after normalization to GAPDH levels. GAPDH is a loading control. Quantification of (A) is shown in (B). Data are shown as mean ± SD from three independent experiments and were analyzed by the Student’s t-test; **P < 0.01; ***P < 0.001 vs. Mock + NC; $$P < 0.01 vs. HSV-1 + NC; #P < 0.05 vs. Mock + OE. (C) Apoptotic cells were quantified by flow cytometry. Data shown are presented as mean ± SD from three independent experiments and were analyzed by the Student’s t-test; ***P < 0.001 vs. Mock + NC; $$$P < 0.001 vs. HSV-1 + NC; ###P < 0.001 vs. Mock + OE. (D) Occludin and claudin 5 protein levels were determined by immunoblotting. Occludin and claudin 5 protein levels in HSV-1-infected cells transfected with pcDNA3.1 or pcDNA3.1-GM130 were normalized to the levels in mock-infected cells transfected with pcDNA3.1 or pcDNA3.1-GM130, respectively, to provide fold changes after normalization to GAPDH levels. GAPDH is a loading control. Quantification of (D) is shown in (E). Data are shown as mean ± SD from three independent experiments and were analyzed by the Student’s t-test; **P < 0.01; ***P < 0.001 vs. Mock + NC; $P < 0.05 vs. HSV-1 + NC; #P < 0.05 vs. Mock + OE. (F) Quantitation of Golgi cisternae lumen (μm) in mock- and HSV-1-infected cells transfected with pcDNA3.1 or pcDNA3.1-GM130. Data represents the average maximum luminal width of Golgi cisternae from three independent experiments. Data are shown as mean ± SD and were analyzed by the Student’s t-test; ***P < 0.001 vs. Mock + NC; $$$P < 0.001 vs. HSV-1 + NC; ##P < 0.01 vs. Mock + OE. Representative electron micrographs of Golgi cisternae in HSV-1-infected cells transfected with pcDNA3.1 (G) and HSV-1-infected cells transfected with pcDNA3.1-GM130 at 36 hpi (H). Representative images are shown from three independent experiments. The black arrowheads indicate Golgi cisternae. The asterisks indicate the nucleus. Scale bar, 1 μm. (I) Growth kinetics of viruses. Twelve hours after transfection, Bend.3 cells transfected with pcDNA3.1 or pcDNA3.1-GM130 were infected with HSV-1 at an MOI of 3. The supernatant was collected at the indicated times post-infection, and viral titer was measured with a plaque formation assay in triplicate. Data shown are presented as mean ± SD from three independent experiments and were analyzed by two-way ANOVA.
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Collected and cropped from Front Mol Neurosci by CiteAb, provided under a CC-BY license
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