Product Citations: 108

Fig.4.D showing Western Blotting from the publication: KLF10 Inhibits TGF-β-Mediated Activation of Hepatic Stellate Cells via Suppression of ATF3 Expression.
Fig.3.D showing Western Blotting from the publication: KLF10 Inhibits TGF-β-Mediated Activation of Hepatic Stellate Cells via Suppression of ATF3 Expression.
Fig.2.D showing Western Blotting from the publication: KLF10 Inhibits TGF-β-Mediated Activation of Hepatic Stellate Cells via Suppression of ATF3 Expression.
Fig.5.A showing Western Blotting from the publication: Intra- and Intertumoral Microglia/Macrophage Infiltration and Their Associated Molecular Signature Is Highly Variable in Canine Oligodendroglioma: A Preliminary Evaluation.
Fig.5.A showing Western Blotting from the publication: Breast cancer cell-derived extracellular vesicles promote CD8+ T cell exhaustion via TGF-β type II receptor signaling.
Fig.4.D showing Western Blotting from the publication: Breast cancer cell-derived extracellular vesicles promote CD8+ T cell exhaustion via TGF-β type II receptor signaling.
Fig.3.A showing Western Blotting from the publication: Arl15 upregulates the TGFβ family signaling by promoting the assembly of the Smad-complex.
Fig.6.D showing Western Blotting in a Mus musculus (House mouse) sample from the publication: Conditional deletion of Nedd4-2 in lung epithelial cells causes progressive pulmonary fibrosis in adult mice.
Fig.3.C showing Western Blotting in a Homo sapiens (Human) sample from the publication: GREB1 induced by Wnt signaling promotes development of hepatoblastoma by suppressing TGFβ signaling.
Fig.6.A showing Western Blotting from the publication: Membrane associated collagen XIII promotes cancer metastasis and enhances anoikis resistance.
Fig.6.C showing Western Blotting from the publication: Membrane associated collagen XIII promotes cancer metastasis and enhances anoikis resistance.
Fig.6.E showing Western Blotting from the publication: Membrane associated collagen XIII promotes cancer metastasis and enhances anoikis resistance.
Fig.2.E showing Western Blotting from the publication: HCC-derived exosomes elicit HCC progression and recurrence by epithelial-mesenchymal transition through MAPK/ERK signalling pathway.
Fig.4.D showing Western Blotting from the publication: TGF-β signalling and PEG10 are mutually exclusive and inhibitory in chondrosarcoma cells.
Fig.4.F showing Western Blotting from the publication: TGF-β signalling and PEG10 are mutually exclusive and inhibitory in chondrosarcoma cells.
Fig.5.C showing Western Blotting from the publication: TGF-β signalling and PEG10 are mutually exclusive and inhibitory in chondrosarcoma cells.
Fig.1.A showing Western Blotting from the publication: Tumor p38MAPK signaling enhances breast carcinoma vascularization and growth by promoting expression and deposition of pro-tumorigenic factors.
Fig.1.G showing Western Blotting from the publication: FAF1 phosphorylation by AKT accumulates TGF-β type II receptor and drives breast cancer metastasis.
Fig.4.C showing Western Blotting from the publication: Genetic depletion and pharmacological targeting of αv integrin in breast cancer cells impairs metastasis in zebrafish and mouse xenograft models.
Fig.5.A showing Western Blotting from the publication: Genetic depletion and pharmacological targeting of αv integrin in breast cancer cells impairs metastasis in zebrafish and mouse xenograft models.
Fig.6.D showing Western Blotting from the publication: A functional genome-wide in vivo screen identifies new regulators of signalling pathways during early Xenopus embryogenesis.
Fig.4.C showing Western Blotting from the publication: A functional genome-wide in vivo screen identifies new regulators of signalling pathways during early Xenopus embryogenesis.
Fig.4.A showing Western Blotting from the publication: A functional genome-wide in vivo screen identifies new regulators of signalling pathways during early Xenopus embryogenesis.
Fig.4.B showing Western Blotting from the publication: A functional genome-wide in vivo screen identifies new regulators of signalling pathways during early Xenopus embryogenesis.
Fig.1.B showing Western Blotting in a Xenopus laevis (African clawed frog) sample from the publication: A functional genome-wide in vivo screen identifies new regulators of signalling pathways during early Xenopus embryogenesis.
26 images found

Protocol for immunofluorescence detection and quantification of phosphorylated SMAD proteins in human blastocysts.

In STAR Protocols on 20 June 2025 by Fallesen, T. & Brumm, A. S.

The transforming growth factor β (TGF-β) signaling superfamily includes NODAL and bone morphogenetic protein (BMP) signaling, which lead to the phosphorylation of different SMAD proteins and regulate key developmental events. Here, we present a protocol for immunofluorescence detection of phosphorylated SMAD proteins combined with other transcription factors in pre-implantation human embryos. We describe steps for segmenting the nuclei in human blastocysts and quantifying their immunofluorescence intensity. This protocol can be adapted to investigate TGF-β superfamily signaling activity in other mammalian embryos or in vitro models of their development. For complete details on the use and execution of this protocol, please refer to Brumm et al.1.
Copyright © 2025 The Authors. Published by Elsevier Inc. All rights reserved.

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Multi-omics reveals global signaling rewiring and identifies Activin A-induced dysregulation of FOS/Activator Protein 1 as a novel target in Fibrodysplasia ossificans progressiva

Preprint on BioRxiv : the Preprint Server for Biology on 21 January 2025 by Wits, M., Gómez-Suárez, N., et al.

Background Fibrodysplasia ossificans progressiva (FOP) is caused by an activating mutation (p.R206H) in the type I BMP receptor ALK2, leading to heterotopic ossification (HO) in soft connective tissues. While aberrant Activin A-induced SMAD signaling is central in FOP pathogenesis, global signaling alterations remain poorly understood. Methods We performed phosphoproteomics, transcriptomics and biochemical analyses in mesenchymal cells (MSCs) overexpressing wild-type ALK2 WT or mutant ALK2 R206H receptors and in induced-MSCs derived from FOP patient iPSCs. Findings were validated in vivo using FOP-like mouse models and in vitro via pharmacological interventions. Results Multi-omics analyses revealed previously unrecognized signaling networks in ALK2 R206H cells, including enhanced MAPK, mTOR, RUNX2 and RHO-mediated mechanotransduction pathways. Notably, we identified dysregulated Activator Protein-1 (AP-1) expression and function as a novel contributor to FOP. AP-1 factors were highly enriched in HO lesions in FOP-like animals. Pharmacological inhibition of AP-1 significantly reduced osteochondrogenic differentiation in vitro. Conclusion This study highlights global signaling dysregulation in FOP and identifies AP-1 as a critical driver and potential therapeutic target for FOP.

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SEMA7A-mediated juxtacrine stimulation of IGFBP-3 upregulates IL-17RB at pancreatic cancer invasive front.

In Cancer Gene Therapy on 1 December 2024 by Chen, Y. I., Tien, S. C., et al.

Tumor invasion is the hallmark of tumor malignancy. The invasive infiltration pattern of tumor cells located at the leading edge is highly correlated with metastasis and unfavorable patient outcomes. However, the regulatory mechanisms governing tumor malignancy at the invasive margin remain unclear. The IL-17B/IL-17RB pathway is known to promote pancreatic cancer invasion and metastasis, yet the specific mechanisms underlying IL-17RB upregulation during invasion are poorly understood. In this study, we unveiled a multistep process for IL-17RB upregulation at the invasive margin, which occurs through direct communication between tumor cells and fibroblasts. Tumor ATP1A1 facilitates plasma membrane expression of SEMA7A, which binds to and induces IGFBP-3 secretion from fibroblasts. The resulting gradient of IGFBP-3 influences the direction and enhances IL-17RB expression to regulate SNAI2 in invasion. These findings highlight the importance of local tumor-fibroblast interactions in promoting cancer cell invasiveness, potentially leading to the development of new therapeutic strategies targeting this communication.
© 2024. The Author(s).

  • WB
  • Cancer Research
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The contribution of the WNT pathway to the therapeutic effects of montelukast in experimental murine airway inflammation induced by ovalbumin and lipopolysaccharide.

In Drug Development Research on 1 April 2024 by Kaya-Yasar, Y., Engin, S., et al.

The wingless/integrase-1 (WNT) pathway involved in the pathogenesis of inflammatory airway diseases has recently generated considerable research interest. Montelukast, a leukotriene receptor antagonist, provides therapeutic benefits in allergic asthma involving eosinophils. We aimed to investigate the role of the WNT pathway in the therapeutic actions of montelukast (MT) in a mixed type of allergic-acute airway inflammation model induced by ovalbumin (OVA) and lipopolysaccharide (LPS) in mice. Female mice were sensitized with intraperitoneal OVA-Al(OH)3 administration in the initiation phase and intranasal OVA followed by LPS administration in the challenge phase. The mice were divided into eight groups: control, asthmatic, and control/asthmatic treated with XAV939 (inhibitor of the canonical WNT pathway), LGK-974 (inhibitor of the secretion of WNT ligands), or MT at different doses. The inhibition of the WNT pathway prevented tracheal 5-HT and bradykinin hyperreactivity, while only the inhibition of the canonical WNT pathway partially reduced 5-HT and bradykinin contractions compared to the inflammation group. Therefore, MT treatment hindered 5-HT and bradykinin hyperreactivity associated with airway inflammation. Furthermore, MT prevented the increases in the phosphorylated GSK-3β and WNT5A levels, which had been induced by airway inflammation, in a dose-dependent manner. Conversely, the MT application caused a further increase in the fibronectin levels, while there was no significant alteration in the phosphorylation of the Smad-2 levels in the isolated lungs of the mice. The MT treatment reversed the increase in the mRNA expression levels of interleukin-17A. An increase in eosinophil and neutrophil counts was observed in bronchoalveolar lavage fluid samples obtained from the mice in the inflammation group, which was hampered by the MT treatment. The inhibition of the WNT pathway did not alter inflammatory cytokine expression or cell infiltration. The WNT pathway mediated the therapeutic effects of MT due to the inhibition of GSK-3β phosphorylation as well as the reduction of WNT5A levels in a murine airway inflammation model.
© 2024 Wiley Periodicals LLC.

  • WB
  • Immunology and Microbiology
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CD44 acts as a coreceptor for cell-specific enhancement of signaling and regulatory T cell induction by TGM1, a parasite TGF-β mimic.

In Proceedings of the National Academy of Sciences of the United States of America on 22 August 2023 by van Dinther, M., Cunningham, K. T., et al.

Long-lived parasites evade host immunity through highly evolved molecular strategies. The murine intestinal helminth, Heligmosomoides polygyrus, down-modulates the host immune system through release of an immunosuppressive TGF-β mimic, TGM1, which is a divergent member of the CCP (Sushi) protein family. TGM1 comprises 5 domains, of which domains 1-3 (D1/2/3) bind mammalian TGF-β receptors, acting on T cells to induce Foxp3+ regulatory T cells; however, the roles of domains 4 and 5 (D4/5) remain unknown. We noted that truncated TGM1, lacking D4/5, showed reduced potency. Combination of D1/2/3 and D4/5 as separate proteins did not alter potency, suggesting that a physical linkage is required and that these domains do not deliver an independent signal. Coprecipitation from cells treated with biotinylated D4/5, followed by mass spectrometry, identified the cell surface protein CD44 as a coreceptor for TGM1. Both full-length and D4/5 bound strongly to a range of primary cells and cell lines, to a greater degree than D1/2/3 alone, although some cell lines did not respond to TGM1. Ectopic expression of CD44 in nonresponding cells conferred responsiveness, while genetic depletion of CD44 abolished enhancement by D4/5 and ablated the ability of full-length TGM1 to bind to cell surfaces. Moreover, CD44-deficient T cells showed attenuated induction of Foxp3 by full-length TGM1, to levels similar to those induced by D1/2/3. Hence, a parasite protein known to bind two host cytokine receptor subunits has evolved a third receptor specificity, which serves to raise the avidity and cell type-specific potency of TGF-β signaling in mammalian cells.

  • Immunology and Microbiology
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KLF10 Inhibits TGF-β-Mediated Activation of Hepatic Stellate Cells via Suppression of ATF3 Expression.

In Int J Mol Sci on 9 August 2023 by Hwang, S., Park, S., et al.

Fig.4.D
Fig.4.D showing Western Blotting from the publication: KLF10 Inhibits TGF-β-Mediated Activation of Hepatic Stellate Cells via Suppression of ATF3 Expression.
  • WB
KLF10 knockdown promotes activation of LX-2 human HSCs via ATF3. LX-2 cells were transfected with siKLF10 for 48 h and further treated with 10 ng/mL TGF-β1 for another 24 h. The chemical chaperone 4-PBA was applied 2 h before TGF-β1 treatment. (A) Cell viability was determined using MTT assays. D... more
KLF10 knockdown promotes activation of LX-2 human HSCs via ATF3. LX-2 cells were transfected with siKLF10 for 48 h and further treated with 10 ng/mL TGF-β1 for another 24 h. The chemical chaperone 4-PBA was applied 2 h before TGF-β1 treatment. (A) Cell viability was determined using MTT assays. Data are expressed as means ± SEM. (B) Levels of α-SMA, fibronectin, and Col1α1 were determined by Western blotting. (C) Collagen accumulation was evaluated by Picro-Sirius red staining. Collagen, red. Scale bar = 100 μm. (D) LX-2 cells were transfected with siKLF10 for 48 h and further treated with 10 ng/mL TGF-β1 for the indicated times. The chemical chaperone 4-PBA was applied 2 h before TGF-β1 treatment. The levels of ATF3, p-SMAD2, p-SMAD3, and SMAD2/3 were determined by Western blotting. GAPDH was used as an internal control. less

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KLF10 Inhibits TGF-β-Mediated Activation of Hepatic Stellate Cells via Suppression of ATF3 Expression.

In Int J Mol Sci on 9 August 2023 by Hwang, S., Park, S., et al.

Fig.3.D
Fig.3.D showing Western Blotting from the publication: KLF10 Inhibits TGF-β-Mediated Activation of Hepatic Stellate Cells via Suppression of ATF3 Expression.
  • WB
KLF10 inhibits TGF-β1-induced activation of LX-2 cells. LX-2 cells were transfected with pcDNA-KLF10 for 48 h and further treated with 10 ng/mL TGF-β1 for another 24 h. (A) Cell viability was determined by MTT assays. (B) Protein levels of α-SMA, fibronectin, and Col1α1 were determined by Western... more
KLF10 inhibits TGF-β1-induced activation of LX-2 cells. LX-2 cells were transfected with pcDNA-KLF10 for 48 h and further treated with 10 ng/mL TGF-β1 for another 24 h. (A) Cell viability was determined by MTT assays. (B) Protein levels of α-SMA, fibronectin, and Col1α1 were determined by Western blotting. (C) Collagen accumulation was evaluated using Picro-Sirius red staining. Collagen, red. Scale bar = 100 μm. (D) LX-2 cells were transfected with pcDNA-KLF10 for 48 h and treated with TGF-β1 for the indicated times. The levels of p-SMAD2, p-SMAD3, SMAD2/3, KLF10, and ATF3 were determined using Western blotting. β-actin was used as an internal control. (E) LX-2 cells were transfected with pcDNA-KLF10 for 48 h and further treated with 10 ng/mL TGF-β1 for another 4 h. mRNA levels of ATF3 were determined by qPCR. Data are expressed as means ± SEM. (F) LX-2 cells were transfected with pcDNA-KLF10 for 48 h and further treated with 10 ng/mL TGF-β1 for 24 h. ATF3 promoter activity was determined by a luciferase assay. * p < 0.05. less

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KLF10 Inhibits TGF-β-Mediated Activation of Hepatic Stellate Cells via Suppression of ATF3 Expression.

In Int J Mol Sci on 9 August 2023 by Hwang, S., Park, S., et al.

Fig.2.D
Fig.2.D showing Western Blotting from the publication: KLF10 Inhibits TGF-β-Mediated Activation of Hepatic Stellate Cells via Suppression of ATF3 Expression.
  • WB
KLF10 knockdown promotes TGF-β-induced activation in human LX-2 HSCs. LX-2 cells were transfected with siKLF10 (100 pmol) for 48 h and further treated with 10 ng/mL TGF-β1 for another 24 h. (A) Cell viability was determined using MTT assays. (B) Protein levels of α-SMA, fibronectin, and Col1α1 we... more
KLF10 knockdown promotes TGF-β-induced activation in human LX-2 HSCs. LX-2 cells were transfected with siKLF10 (100 pmol) for 48 h and further treated with 10 ng/mL TGF-β1 for another 24 h. (A) Cell viability was determined using MTT assays. (B) Protein levels of α-SMA, fibronectin, and Col1α1 were determined by Western blotting. (C) Collagen accumulation was evaluated by Picro-Sirius red staining. Collagen, red. Scale bars = 100 μm. (D) LX-2 cells transfected with siKLF10 were treated with TGF-β1 for the indicated time periods. The levels of p-SMAD2, p-SMAD3, SMAD2/3, KLF10, and ATF3 were determined by Western blotting. β-actin was used as an internal control. The protein band of Western blotting was quantified by ImageJ and normalized using β-actin. (E) LX-2 cells were transfected with siKLF10 (100 pmol) for 48 h and further treated with 10 ng/mL TGF-β1 for another 4 h. The mRNA levels of ATF3 were determined by qPCR. Data are expressed as means ± SEM. (F) LX-2 cells were transfected with siKLF10 (100 pmol) for 48 h and then treated with 10 ng/mL TGF-β1 for 24 h. ATF3 promoter activity was determined using a luciferase assay. * p < 0.05. less

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Intra- and Intertumoral Microglia/Macrophage Infiltration and Their Associated Molecular Signature Is Highly Variable in Canine Oligodendroglioma: A Preliminary Evaluation.

In Vet Sci on 19 June 2023 by Toedebusch, R. G., Wei, N. W., et al.

Fig.5.A
Fig.5.A showing Western Blotting from the publication: Intra- and Intertumoral Microglia/Macrophage Infiltration and Their Associated Molecular Signature Is Highly Variable in Canine Oligodendroglioma: A Preliminary Evaluation.
  • WB
TGFβ and galectin-3 signaling may be relevant in canine oligodendroglioma. (A) Representative immunoblots for proteins within the canonical transforming growth factor β (TGFβ) pathway and galectin-3 (GAL-3) in normal canine brain (n = 3) and HGG homogenates (n = 4). Relative protein expression of... more
TGFβ and galectin-3 signaling may be relevant in canine oligodendroglioma. (A) Representative immunoblots for proteins within the canonical transforming growth factor β (TGFβ) pathway and galectin-3 (GAL-3) in normal canine brain (n = 3) and HGG homogenates (n = 4). Relative protein expression of (B) TGFβ targets or (C) GAL-3 (p = 0.0608) was not different between normal brain (n = 3) and HGG homogenate (n = 4). Comparisons based on unpaired t-test; bars represent group mean with standard error of the mean. (D) Representative immunofluorescence staining for GAL-3 in HGG. Multifocal areas of robust cytoplasmic GAL-3 IR were observed to co-localize with OLIG2 IR (top panel). Serial sections of tumor areas rich in IBA-1 IR had weak GAL-3 IR without co-localization with IBA-1-positive cells (bottom panel). Scale bars, 5 μm. less

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Breast cancer cell-derived extracellular vesicles promote CD8+ T cell exhaustion via TGF-β type II receptor signaling.

In Nat Commun on 1 August 2022 by Xie, F., Zhou, X., et al.

Fig.5.A
Fig.5.A showing Western Blotting from the publication: Breast cancer cell-derived extracellular vesicles promote CD8+ T cell exhaustion via TGF-β type II receptor signaling.
  • WB
EV-TβRII promotes breast cancer stemness and metastatic outgrowth.a Immunoblot analysis of total cells lysates from MCF7 cells (upper panel) or 4T07 cells (lower panel) pre-treated for 24 h with TβRII+ EVs or TβRII− EVs (40 μg) derived from MDA-MB-231 cells, and stimulated with or without TGF-β (... more
EV-TβRII promotes breast cancer stemness and metastatic outgrowth.a Immunoblot analysis of total cells lysates from MCF7 cells (upper panel) or 4T07 cells (lower panel) pre-treated for 24 h with TβRII+ EVs or TβRII− EVs (40 μg) derived from MDA-MB-231 cells, and stimulated with or without TGF-β (5 ng/ml) for 1 h as indicated. b Immunoblot analysis of total cell lysate of MCF10A-RAS cells pre-treated for 48 h with TβRII+ (RII+) EVs or TβRII− (RII−) EVs (40 μg) derived from MDA-MB-231 cells, and treated with or without SB431542 (10 μM) for 48 h as indicated. c Immunofluorescence and DAPI staining of HaCaT cells pre-treated for 48 h with TβRII+ (RII+) EVs or TβRII− (RII−) EVs (40 μg) derived from MDA-MB-231 cells, and treated with or without TGF-β (5 ng/ml) and SB431542 (10 μM) for 48 h as indicated. Scale bars, 20 μm. d MCF10A-RAS cells pre-treated with control EVs, TβRII+ (RII+) EVs, or TβRII− (RII−) EVs (40 μg) derived from MDA-MB-231 cells were analyzed in a tumor sphere assay. Quantification of the number of tumor spheres per 5 × 103 seeded cells (left panel) and pictures show representative images of tumor spheres (right panel). Scale bar, 2 mm. e FACS analysis (left panel) and Quantification (right panel) of the CD44high/CD24low population in MCF10A-RAS cells treated with Co.EVs, TβRII+ or TβRII− EVs (40 μg) for 48 h. f Cytotoxic crystal violet staining (left) and dose-response curves (right) to paclitaxel (PTX) of MDA-MB-231 cells pre-incubated with Co.EVs, TβRII+ or TβRII− EVs (40 μg) for 48 h. The dose is presented on the x axis, while cell viability (% vs control) is presented on the y axis. g Cytotoxic crystal violet staining (left) and dose-response curves (right) to Doxorubicin (Doxo) of MDA-MB-231 cells pre-incubated with Co.EVs, TβRII+ or TβRII− EVs (40 μg) for 48 h. The dose is presented on the x axis, while cell viability (% vs control) is presented on the y axis. h–k EVs administration and experimental analysis in vivo: 4T07 cells pre-treated with Co.EVs, TβRII+ or TβRII− EVs (40 μg) were tail vein-injected into nude mice (n = 8 mice per group). Mice were also given injection (into the tail vein) of corresponding EVs (50 μg per mouse every other day) before the tumor injection h; the percentage of metastasis-free mice in each experimental group followed in time i; Lung metastasis was measured by BLI. Normalized photon flux (left panel), representative images (right panel) in each experimental group followed in time j; Representative images of bright view (upper), BLI signal (scale bars, 2 mm, middle upper), HE (scale bars, 1 mm, middle lower) and IHC staining (scale bars, 2 μm, lower) in each group k. ns not significant (p > 0.05) and *p < 0.05 (unpaired two-tailed Student’s t test d, e or two-way ANOVA f, g, j). Data are analyzed from three independent experiments and shown as mean ± SD d, e, f, g, j. Source data are provided as a Source Data file. less

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Breast cancer cell-derived extracellular vesicles promote CD8+ T cell exhaustion via TGF-β type II receptor signaling.

In Nat Commun on 1 August 2022 by Xie, F., Zhou, X., et al.

Fig.4.D
Fig.4.D showing Western Blotting from the publication: Breast cancer cell-derived extracellular vesicles promote CD8+ T cell exhaustion via TGF-β type II receptor signaling.
  • WB
EV-TβRII mediates the TEVs-induced SMAD activation in recipient cells.a Schematic diagram (left panel), confocal microscopy (middle panel) and quantification (right panel) of MCF7 cells incubated with EVs derived from MDA-MB-231 cells stably expressing TβRII-GFP for indicated times. Scale bar, 20... more
EV-TβRII mediates the TEVs-induced SMAD activation in recipient cells.a Schematic diagram (left panel), confocal microscopy (middle panel) and quantification (right panel) of MCF7 cells incubated with EVs derived from MDA-MB-231 cells stably expressing TβRII-GFP for indicated times. Scale bar, 20 μm. b Schematic diagram (left panel), FACS analysis (middle panel) and quantification (right panel) of MCF7 cells incubated with MDA-MB-231 cells stably expressing TβRII-GFP for indicated times. c Transcriptional analysis of TGF-β-induced (2.5 ng/ml for 16 hrs) CAGA12-Luc response in TβRII-deficient mink lung epithelial DR26 cells pre-treated for 24 h with empty liposome (as a control; Co.EVs), TβRII+ (RII+) EVs (40 μg) derived from untransfected MDA-MB-231 cells or TβRII−(RII−) EVs (40 μg) derived from TβRII knockout MDA-MB-231 cells. d Immunoblot analysis of total lysates of DR26 cells pre-incubated with empty liposome (as a control; Co.EVs) or EVs (40 μg) derived from TβRII+ (RII+) or TβRII−(RII−) MDA-MB-231 cells, and stimulated with TGF-β (2.5 ng/ml) for 1 h as indicated. e Transcriptional analysis of CAGA12-Luc response in TβRII-deficient DR26 cells incubated with empty liposome (as a control; Co.EVs) or EVs (40 μg) derived from TβRII+ (RII+) MDA-MB-231 cells (MDA EVs) and treated with control DMSO, SB431542 (10 μM) or LY-364947 (10 μM) as indicated. f Immunofluorescence and DAPI staining of CAF cells pre-incubated with Co.EVs, TβRII+ or TβRII− EVs (40 μg) and treated with control DMSO, SB431542 (10 μM) or LY-364947 (10 μM) as indicated. Scale bar, 20 μm. g Transcriptional analysis of CAGA12-Luc response in HEK293T cells as indicated pre-treated for 24 h with EVs (40 μg) derived from TβRII+ (RII+) or TβRII−(RII−) MDA-MB-231 cells. h qPCR analysis in MCF7 cells pre-incubated for 48 h with Co.EVs, TβRII+ or TβRII− EVs (40 μg), followed by no stimulation (−) or stimulation (+) for 16 h with TGF-β (2.5 ng/ml). Relative mRNA levels are shown as a heatmap. *p < 0.05 (two-tailed Student’s t test (a, b, c, e, g)). Data are analyzed of three independent experiments and shown as mean + SD (a, b, c, e, g). Source data are provided as a Source Data file. less

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Arl15 upregulates the TGFβ family signaling by promoting the assembly of the Smad-complex.

In Elife on 14 July 2022 by Shi, M., Tie, H. C., et al.

Fig.3.A
Fig.3.A showing Western Blotting from the publication: Arl15 upregulates the TGFβ family signaling by promoting the assembly of the Smad-complex.
  • WB
Arl15-GTP indirectly interacts with Smad2 via Smad4.HEK293T cells under the normal culture condition were used. (a) The anti-Smad2/3 mAb primarily detects endogenous Smad2 in HEK293T cells. Cells were subjected to GL2, Smad2, or Smad3 siRNA knockdown, and the resulting cell lysates were blotted f... more
Arl15-GTP indirectly interacts with Smad2 via Smad4.HEK293T cells under the normal culture condition were used. (a) The anti-Smad2/3 mAb primarily detects endogenous Smad2 in HEK293T cells. Cells were subjected to GL2, Smad2, or Smad3 siRNA knockdown, and the resulting cell lysates were blotted for indicated proteins. Ratios of the intensity of Smad2/3 band to that of GAPDH band are displayed below. (b) The GTP-mutant form of Arl15 pulls down more exogenously expressed Smad2 when Smad4 was co-expressed. Bead-immobilized GST-fusion proteins were incubated with cell lysates expressing indicated proteins, and pull-downs and 1% cell lysate inputs were immunoblotted against Smad2 (anti-Smad2/3 mAb) and Smad4. In the middle panel, the same blot was sequentially blotted for Smad2 (upper blot) followed by Smad4 (lower blot). 1, GFP-Smad2; 2, GST-Arl15 (AL or TN); 3, GST; 4, GFP-Smad4; #, endogenous Smad2; *, the weak band of GFP-Smad2 that was pulled down without co-expression of Smad4. The normalized pull-down of GFP-Smad2, calculated by the ratio of the intensity of the pull-down band to that of 1% cell lysate input band, is plotted in the right panel. Loading of fusion proteins is shown by Coomassie staining. Molecular weights (in kDa) are labeled in immunoblots and gels.Figure 3—figure supplement 1—source data 1.Uncropped gel and blot images for Figure 3—figure supplement 1.The organization of the figure is similar to that of Figure 1—source data 1.Uncropped gel and blot images for Figure 3—figure supplement 1.The organization of the figure is similar to that of Figure 1—source data 1. less

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Conditional deletion of Nedd4-2 in lung epithelial cells causes progressive pulmonary fibrosis in adult mice.

In Nat Commun on 24 April 2020 by Duerr, J., Leitz, D. H. W., et al.

Fig.6.D
Fig.6.D showing Western Blotting in a Mus musculus (House mouse) sample from the publication: Conditional deletion of Nedd4-2 in lung epithelial cells causes progressive pulmonary fibrosis in adult mice.
  • WB
  • Mus musculus (House mouse)
Conditional deletion of Nedd4-2 in lung epithelial cells causes increased levels of TGFβ and exaggerated Smad2/3 signaling.a Levels of active TGFβ in whole lung of conditional Nedd4-2−/− mice and littermate controls after 0.5 (n = 4/group), 2 (n = 4/group), 3 (n = 5/group), and 4 months (n = 6/gr... more
Conditional deletion of Nedd4-2 in lung epithelial cells causes increased levels of TGFβ and exaggerated Smad2/3 signaling.a Levels of active TGFβ in whole lung of conditional Nedd4-2−/− mice and littermate controls after 0.5 (n = 4/group), 2 (n = 4/group), 3 (n = 5/group), and 4 months (n = 6/group) of doxycycline induction. b, c mRNA expression levels of the mesenchymal marker genes Vim and Fn1 (n = 9/group) (b) and alveolar type 2 (AT2) cell markers Sftpc (n = 9/group) and Sftpd (n = 6/group) (c) of whole lungs from conditional Nedd4-2−/− and control mice after 3 months of doxycycline induction. d–i AT2 cells isolated from conditional Nedd4-2−/− and control mice induced with doxycycline for 2 weeks were pretreated with 1 ng/ml TGFβ and analyzed for phosphorylated Smad2/3 (pSmad2/3) and transcript levels of TGFβ-inducible genes. d–f Representative Western blot (d) and densitometric analysis of pSmad2 (e) and pSmad3 (f) at baseline and after 1–3 h of TGFβ stimulation. g–i mRNA levels of Serpine1 (g), Smad7 (h), and Skil (i) after the indicated time of TGFβ stimulation. All in vitro data are derived from 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared to controls. Statistical analysis was performed using two-tailed Mann–Whitney test in a, b, two-tailed unpaired t test in c, and two-way ANOVA with Sidac’s post hoc test in e–i. Data are shown as mean ± S.E.M. Source data are provided in the Source Data file. less

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GREB1 induced by Wnt signaling promotes development of hepatoblastoma by suppressing TGFβ signaling.

In Nat Commun on 28 August 2019 by Matsumoto, S., Yamamichi, T., et al.

Fig.3.C
Fig.3.C showing Western Blotting in a Homo sapiens (Human) sample from the publication: GREB1 induced by Wnt signaling promotes development of hepatoblastoma by suppressing TGFβ signaling.
  • WB
  • Homo sapiens (Human)
GREB1 forms a complex with Smad2/3. a Schematic representation of the GREB1 and GREB1-ΔNLS (310–319) are shown. X293T cells expressing the indicated proteins were fixed and stained with anti-FLAG antibody, phalloidin, and Hoechst33342. b Lysates of X293T cells expressing the indicated proteins we... more
GREB1 forms a complex with Smad2/3. a Schematic representation of the GREB1 and GREB1-ΔNLS (310–319) are shown. X293T cells expressing the indicated proteins were fixed and stained with anti-FLAG antibody, phalloidin, and Hoechst33342. b Lysates of X293T cells expressing the indicated proteins were immunoprecipitated with anti-GFP antibody, and the immunoprecipitates were probed with the indicated antibodies. c Lysates of HepG2 cells were immunoprecipitated with anti-Smad2/3 antibody and the immunoprecipitates were probed with the indicated antibodies. d Schematic representation of the deletion mutants of Smad2 used in this study is shown. Lysates of X293T cells expressing HA-FLAG-GREB1 and GFP-Smad2 mutants were immunoprecipitated with anti-GFP antibody and the immunoprecipitates were probed with the indicated antibodies. e Lysates of HepG2 cells were precipitated with recombinant GST, GST-Smad2/MH1, or GST-Smad2/MH2 proteins and the precipitates were probed with the indicated antibodies. f Schematic representation of three deletion mutants (N, M, and C) of GFP-GREB1 is shown. HepG2 cells expressing deletion mutants of GFP-GREB1 were fixed and stained with anti-GFP antibody. g Lysates of X293T cells expressing FLAG-Smad3 and GFP-GREB1 mutants were immunoprecipitated with anti-GFP antibody and the immunoprecipitates were probed with the indicated antibodies. Scale bars in a and f, 20 μm less

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Membrane associated collagen XIII promotes cancer metastasis and enhances anoikis resistance.

In Breast Cancer Res on 1 October 2018 by Zhang, H., Fredericks, T., et al.

Fig.6.A
Fig.6.A showing Western Blotting from the publication: Membrane associated collagen XIII promotes cancer metastasis and enhances anoikis resistance.
  • WB
Collagen XIII enhances activation of the TGF-β pathway. a Western blot analyzed TGF-β-induced Smad phosphorylation in control and Col13−/− MDA-MB-231 cells. b Bar graph quantifying TGF-β-induced TPA response elements (TREs)-driven luciferase activity in control and Col13−/− MDA-MB-231; n = 3; ** ... more
Collagen XIII enhances activation of the TGF-β pathway. a Western blot analyzed TGF-β-induced Smad phosphorylation in control and Col13−/− MDA-MB-231 cells. b Bar graph quantifying TGF-β-induced TPA response elements (TREs)-driven luciferase activity in control and Col13−/− MDA-MB-231; n = 3; ** p < 0.01. c Western blot analyzed TGF-β-induced Smad phosphorylation in control and Col13-expressing MCF-10A cells. d Bar graph showing TGF-β-induced TRE-driven luciferase activity in control and Col13-expressing MCF-10A cells; n = 4, *** p < 0.001. e AIIB2 treatment reduced the level of TGF-β-induced Smad2/3 phosphorylation in Col13-expressing MCF-10A cells. Western bolt images (left) and quantification (right); n = 3; *** p <0.001; n.s., no significance less

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Membrane associated collagen XIII promotes cancer metastasis and enhances anoikis resistance.

In Breast Cancer Res on 1 October 2018 by Zhang, H., Fredericks, T., et al.

Fig.6.C
Fig.6.C showing Western Blotting from the publication: Membrane associated collagen XIII promotes cancer metastasis and enhances anoikis resistance.
  • WB
Collagen XIII enhances activation of the TGF-β pathway. a Western blot analyzed TGF-β-induced Smad phosphorylation in control and Col13−/− MDA-MB-231 cells. b Bar graph quantifying TGF-β-induced TPA response elements (TREs)-driven luciferase activity in control and Col13−/− MDA-MB-231; n = 3; ** ... more
Collagen XIII enhances activation of the TGF-β pathway. a Western blot analyzed TGF-β-induced Smad phosphorylation in control and Col13−/− MDA-MB-231 cells. b Bar graph quantifying TGF-β-induced TPA response elements (TREs)-driven luciferase activity in control and Col13−/− MDA-MB-231; n = 3; ** p < 0.01. c Western blot analyzed TGF-β-induced Smad phosphorylation in control and Col13-expressing MCF-10A cells. d Bar graph showing TGF-β-induced TRE-driven luciferase activity in control and Col13-expressing MCF-10A cells; n = 4, *** p < 0.001. e AIIB2 treatment reduced the level of TGF-β-induced Smad2/3 phosphorylation in Col13-expressing MCF-10A cells. Western bolt images (left) and quantification (right); n = 3; *** p <0.001; n.s., no significance less

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Membrane associated collagen XIII promotes cancer metastasis and enhances anoikis resistance.

In Breast Cancer Res on 1 October 2018 by Zhang, H., Fredericks, T., et al.

Fig.6.E
Fig.6.E showing Western Blotting from the publication: Membrane associated collagen XIII promotes cancer metastasis and enhances anoikis resistance.
  • WB
Collagen XIII enhances activation of the TGF-β pathway. a Western blot analyzed TGF-β-induced Smad phosphorylation in control and Col13−/− MDA-MB-231 cells. b Bar graph quantifying TGF-β-induced TPA response elements (TREs)-driven luciferase activity in control and Col13−/− MDA-MB-231; n = 3; ** ... more
Collagen XIII enhances activation of the TGF-β pathway. a Western blot analyzed TGF-β-induced Smad phosphorylation in control and Col13−/− MDA-MB-231 cells. b Bar graph quantifying TGF-β-induced TPA response elements (TREs)-driven luciferase activity in control and Col13−/− MDA-MB-231; n = 3; ** p < 0.01. c Western blot analyzed TGF-β-induced Smad phosphorylation in control and Col13-expressing MCF-10A cells. d Bar graph showing TGF-β-induced TRE-driven luciferase activity in control and Col13-expressing MCF-10A cells; n = 4, *** p < 0.001. e AIIB2 treatment reduced the level of TGF-β-induced Smad2/3 phosphorylation in Col13-expressing MCF-10A cells. Western bolt images (left) and quantification (right); n = 3; *** p <0.001; n.s., no significance less

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HCC-derived exosomes elicit HCC progression and recurrence by epithelial-mesenchymal transition through MAPK/ERK signalling pathway.

In Cell Death Dis on 1 May 2018 by Chen, L., Guo, P., et al.

Fig.2.E
Fig.2.E showing Western Blotting from the publication: HCC-derived exosomes elicit HCC progression and recurrence by epithelial-mesenchymal transition through MAPK/ERK signalling pathway.
  • WB
Incubation of HLE cells with MHCC97H-derived exosomes promotes EMT through the MAPK/ERK pathway.a Immunofluorescence microscopy of EMT markers in HLE cells treated with or without MHCC97H-derived exosomes for 24 h. b Western blot analysis of EMT markers in HLE cells treated with or without MHCC97... more
Incubation of HLE cells with MHCC97H-derived exosomes promotes EMT through the MAPK/ERK pathway.a Immunofluorescence microscopy of EMT markers in HLE cells treated with or without MHCC97H-derived exosomes for 24 h. b Western blot analysis of EMT markers in HLE cells treated with or without MHCC97H-derived exosomes for 24 h. c Western blot analysis of EMT promoters (ZEB1, ZEB2 and Slug) and MET promoter OVOL1 in HLE cells treated with or without MHCC97H-derived exosomes for 24 h. d Western blot analysis of EMT markers of HLE cells with exposure to MHCC97H-derived exosomes for different durations. e Western blot analysis of phosphorylated and total Erk1/2, Akt and Smad2/3 in HLE cells treated with or without MHCC97H-derived exosomes for 24 h. f Western blot analysis of EMT markers and phosphorylated and total Erk1/2 in HLE cells, exosome-treated HLE cells and exosome-treated HLE cells when cultured with the ERK inhibitor PD98059. Abbreviation: Exo exosome. All experiments were repeated at least three times less

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TGF-β signalling and PEG10 are mutually exclusive and inhibitory in chondrosarcoma cells.

In Sci Rep on 18 October 2017 by Shinohara, N., Maeda, S., et al.

Fig.4.D
Fig.4.D showing Western Blotting from the publication: TGF-β signalling and PEG10 are mutually exclusive and inhibitory in chondrosarcoma cells.
  • WB
PEG10 is negatively regulated by TGF-β signalling. (a) Expression of PEG10 in C28/I2 cells was examined by RT-qPCR at 2 days after application of TGF-β1 (1 ng/ml) or BMP-6 (100 ng/ml) (n = 3). (b) Expression of PEG10 in Hs 819.T cells was examined by RT-qPCR at 7 days after application of TGF-β1 ... more
PEG10 is negatively regulated by TGF-β signalling. (a) Expression of PEG10 in C28/I2 cells was examined by RT-qPCR at 2 days after application of TGF-β1 (1 ng/ml) or BMP-6 (100 ng/ml) (n = 3). (b) Expression of PEG10 in Hs 819.T cells was examined by RT-qPCR at 7 days after application of TGF-β1 (1 ng/ml) or BMP-6 (100 ng/ml) (n = 3). (c) Expression of PEG10 in SW1353 cells was examined by RT-qPCR at 2 days after application of TGF-β1 or BMP-6 at the indicated concentrations (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001 (c,d); n.s., not significant. (d) Expression of PEG10 protein in SW1353 cells was examined by immunoblotting at the indicated time points after application of TGF-β1 (1 ng/ml) or BMP-6 (100 ng/ml). Blots were cropped and the full-length blots are presented in Supplementary Figure S5. Band intensities of PEG10 and tubulin at 2 days (lanes 11–13) were quantified using ImageJ software (e). The intensity of PEG10 was normalised to that of tubulin. (f,g) Chondrosarcoma cell lines were treated with DMSO (D, 0.01%), SB431542 (S, 1 μM), or LDN193189 (L, 0.1 μM) in serum-free medium containing ITS supplement overnight, followed by addition of TGF-β1 (1 ng/ml) or BMP-6 (100 ng/ml) for 30 min and were then immunoblotted using the indicated antibodies. Tubulin served as a loading control. Blots were cropped and the full-length blots are presented in Supplementary Figure S5. Bands intensities of PEG10 and tubulin (lanes 1–3 and 8–10) were quantified using ImageJ software (g). The band intensity of PEG10 was normalised to that of tubulin. less

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TGF-β signalling and PEG10 are mutually exclusive and inhibitory in chondrosarcoma cells.

In Sci Rep on 18 October 2017 by Shinohara, N., Maeda, S., et al.

Fig.4.F
Fig.4.F showing Western Blotting from the publication: TGF-β signalling and PEG10 are mutually exclusive and inhibitory in chondrosarcoma cells.
  • WB
PEG10 is negatively regulated by TGF-β signalling. (a) Expression of PEG10 in C28/I2 cells was examined by RT-qPCR at 2 days after application of TGF-β1 (1 ng/ml) or BMP-6 (100 ng/ml) (n = 3). (b) Expression of PEG10 in Hs 819.T cells was examined by RT-qPCR at 7 days after application of TGF-β1 ... more
PEG10 is negatively regulated by TGF-β signalling. (a) Expression of PEG10 in C28/I2 cells was examined by RT-qPCR at 2 days after application of TGF-β1 (1 ng/ml) or BMP-6 (100 ng/ml) (n = 3). (b) Expression of PEG10 in Hs 819.T cells was examined by RT-qPCR at 7 days after application of TGF-β1 (1 ng/ml) or BMP-6 (100 ng/ml) (n = 3). (c) Expression of PEG10 in SW1353 cells was examined by RT-qPCR at 2 days after application of TGF-β1 or BMP-6 at the indicated concentrations (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001 (c,d); n.s., not significant. (d) Expression of PEG10 protein in SW1353 cells was examined by immunoblotting at the indicated time points after application of TGF-β1 (1 ng/ml) or BMP-6 (100 ng/ml). Blots were cropped and the full-length blots are presented in Supplementary Figure S5. Band intensities of PEG10 and tubulin at 2 days (lanes 11–13) were quantified using ImageJ software (e). The intensity of PEG10 was normalised to that of tubulin. (f,g) Chondrosarcoma cell lines were treated with DMSO (D, 0.01%), SB431542 (S, 1 μM), or LDN193189 (L, 0.1 μM) in serum-free medium containing ITS supplement overnight, followed by addition of TGF-β1 (1 ng/ml) or BMP-6 (100 ng/ml) for 30 min and were then immunoblotted using the indicated antibodies. Tubulin served as a loading control. Blots were cropped and the full-length blots are presented in Supplementary Figure S5. Bands intensities of PEG10 and tubulin (lanes 1–3 and 8–10) were quantified using ImageJ software (g). The band intensity of PEG10 was normalised to that of tubulin. less

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TGF-β signalling and PEG10 are mutually exclusive and inhibitory in chondrosarcoma cells.

In Sci Rep on 18 October 2017 by Shinohara, N., Maeda, S., et al.

Fig.5.C
Fig.5.C showing Western Blotting from the publication: TGF-β signalling and PEG10 are mutually exclusive and inhibitory in chondrosarcoma cells.
  • WB
Canonical TGF-β/BMP signalling is mildly suppressed by PEG10. (a,b) The 9xCAGA or BRE luciferase reporter plasmid together with a Renilla reporter were transfected with or without a Peg10 expression vector followed, 8 h later, by stimulation with TGF-β1 (1 ng/ml) or BMP-6 (100 ng/ml) overnight, r... more
Canonical TGF-β/BMP signalling is mildly suppressed by PEG10. (a,b) The 9xCAGA or BRE luciferase reporter plasmid together with a Renilla reporter were transfected with or without a Peg10 expression vector followed, 8 h later, by stimulation with TGF-β1 (1 ng/ml) or BMP-6 (100 ng/ml) overnight, respectively. Firefly reporter activity was normalised to Renilla activity (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001. (c) SW1353 chondrosarcoma cells were transfected with control or PEG10 siRNAs followed, 12 h later, by stimulation for 1 h with TGF-β1 (1 ng/ml) or BMP-6 (100 ng/ml). Cells were then immunoblotted using the indicated antibodies. Tubulin served as a loading control. Blots were cropped and the full-length blots are presented in Supplementary Figure S6. less

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Tumor p38MAPK signaling enhances breast carcinoma vascularization and growth by promoting expression and deposition of pro-tumorigenic factors.

In Oncotarget on 22 September 2017 by Limoge, M., Safina, A., et al.

Fig.1.A
Fig.1.A showing Western Blotting from the publication: Tumor p38MAPK signaling enhances breast carcinoma vascularization and growth by promoting expression and deposition of pro-tumorigenic factors.
  • WB
p38MAPK contributes to breast carcinoma invasion and metastasis(A) Immunoblotting of whole-cell lysates from breast cancer MDA-MB-231 cells transduced with empty-vector control (EGFP) or Flag-tagged p38MAPK-AGF (dn-p38). Cells were treated with 2 ng/mL TGF-β1 for the indicated times. (B) Invasion... more
p38MAPK contributes to breast carcinoma invasion and metastasis(A) Immunoblotting of whole-cell lysates from breast cancer MDA-MB-231 cells transduced with empty-vector control (EGFP) or Flag-tagged p38MAPK-AGF (dn-p38). Cells were treated with 2 ng/mL TGF-β1 for the indicated times. (B) Invasion of MDA-MB-231 cells tested using Matrigel-covered transwells. Assays were done in triplicate and repeated at least twice. (C) Lung surface colonies of EGFP and dn-p38 MDA-MB-231 cells after tail-vein injection of tumor cells into female SCID mice, 6 mice/group). **, P<0.01 less

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FAF1 phosphorylation by AKT accumulates TGF-β type II receptor and drives breast cancer metastasis.

In Nat Commun on 26 April 2017 by Xie, F., Jin, K., et al.

Fig.1.G
Fig.1.G showing Western Blotting from the publication: FAF1 phosphorylation by AKT accumulates TGF-β type II receptor and drives breast cancer metastasis.
  • WB
FAF1 specifically associates with TβRII and inhibits TGF-β signalling.(a) Bioluminescent imaging (BLI) of representative mice from each group injected into the left heart ventricle with control (Co.) or MDA-MB-231 breast cancer cells stably depleted of TβRII (shTβRII). Images were captured at wee... more
FAF1 specifically associates with TβRII and inhibits TGF-β signalling.(a) Bioluminescent imaging (BLI) of representative mice from each group injected into the left heart ventricle with control (Co.) or MDA-MB-231 breast cancer cells stably depleted of TβRII (shTβRII). Images were captured at week 7. Dorsal images are shown. The BLI signal of every mouse in each experimental group is shown in the middle panel. The percentage of bone metastasis-free mice (right panel) in each experimental group is given. (b) Immunoblot (IB) analysis of TβRII protein levels in parental (P) and metastatic (M) variant of MDA-MB-231 and A549 cells (left panel). Quantitative PCR with reverse transcription analyses of TβRII in parental and metastatic cells are shown (right panel). Data are presented as the mean±s.d. (c) MDA-MB-231 cells were treated with MG132 (25 μM), lactacystin (30 μM), bafilomycin A1 (1 μM), NH4Cl (25 mM) or chloroquine (100 mM) for 5 h and collected for IB analysis. Actin IB is included as a loading control. (d) Mass spectrometry results of TβRII-Flag immunoprecipitants in the absence (sample A) and presence (sample B) of NH4Cl (25 mM) identified FAF1 as a binding partner; exemplified peptide is shown in the right panel. (e) IB of the input and immunoprecipitants (IPs) derived from MCF10A-RAS cells treated with NH4Cl (25 mM) as indicated; 5% of the total cell lysate was loaded as input. NS, non-specific antibody. (f) The CAGA12-Luc SMAD-dependent transcriptional response in HEK293T cells transfected with FAF1 wt/UBX-deleted mutant (▴UBX) or shFAF1, as indicated, and treated with TGF-β (5 ng ml−1) overnight. The data are presented as the mean±s.d. of three independent sets of experiments. (g,h) The IBs of total cell lysate (TCL) and anti-SMAD2/3 immunoprecipitants derived from control and FAF1 stable depletion (shFAF1#1 and shFAF1#2) (g) or Myc-FAF1 fl (full length)/UBX-deleted mutant stably expressing (h) MDA-MB-231 cells treated with ligand at the indicated time points. (i) Oncomine box plots of the FAF1 expression levels in multiple human advanced cancers versus normal tissues; P<0.01. (j) Kaplan–Meier curves of The Cancer Genome Atlas database showing that the metastasis-free survival of individuals positively correlated with FAF1 expression by the log-rank test (P<0.01). less

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Genetic depletion and pharmacological targeting of αv integrin in breast cancer cells impairs metastasis in zebrafish and mouse xenograft models.

In Breast Cancer Res on 25 February 2015 by Li, Y., Drabsch, Y., et al.

Fig.4.C
Fig.4.C showing Western Blotting from the publication: Genetic depletion and pharmacological targeting of αv integrin in breast cancer cells impairs metastasis in zebrafish and mouse xenograft models.
  • WB
αv integrin and transforming growth factor (TGF)-β signaling interaction in breast cancer progression. (A) MDA-MB-231 cells were cultured with or without TGF-β for the indicated times, and RNA was isolated for αv integrin expression analysis by qRT-PCR. (B) Left, transwell assay. Control and knoc... more
αv integrin and transforming growth factor (TGF)-β signaling interaction in breast cancer progression. (A) MDA-MB-231 cells were cultured with or without TGF-β for the indicated times, and RNA was isolated for αv integrin expression analysis by qRT-PCR. (B) Left, transwell assay. Control and knockdown MDA-MB-231 cells were treated with or without 5 ng/ml TGF-β for 16 h and migrated cells were stained. Right, average cell numbers of migrated cells ± SD. (C) Immunoblot analysis of phosphorylated Smad2 and Smad3 (p-Smad2 and 3) and total Smad2 and 3 in control or αv integrin knockdown MDA-MB-231 cells. Cells were treated with or without 5 ng/ml TGF-β for 2 h. (D) Knockdown of αv integrin in MDA-MB-231 cells inhibits expression of TGF-β target genes upon 6 h TGF-β treatment. mRNA levels of PAI-1, CTGF, and IL-11 were assessed by real-time quantitative PCR, and normalized to GAPDH expression. less

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Genetic depletion and pharmacological targeting of αv integrin in breast cancer cells impairs metastasis in zebrafish and mouse xenograft models.

In Breast Cancer Res on 25 February 2015 by Li, Y., Drabsch, Y., et al.

Fig.5.A
Fig.5.A showing Western Blotting from the publication: Genetic depletion and pharmacological targeting of αv integrin in breast cancer cells impairs metastasis in zebrafish and mouse xenograft models.
  • WB
GLPG0187 suppresses breast tumor invasion and metastasis in zebrafish. (A) MDA-MB-231 cells were treated with vehicle, 0.5 ng/ml or 1 ng/ml of GLPG0187 for 48 h with or without 5 ng/ml transforming growth factor (TGF)-β for 2 h and immunoblotted as shown. (B) MDA-MB-231 cells were treated with ve... more
GLPG0187 suppresses breast tumor invasion and metastasis in zebrafish. (A) MDA-MB-231 cells were treated with vehicle, 0.5 ng/ml or 1 ng/ml of GLPG0187 for 48 h with or without 5 ng/ml transforming growth factor (TGF)-β for 2 h and immunoblotted as shown. (B) MDA-MB-231 cells were treated with vehicle, 0.5 ng/ml or 1 ng/ml of GLPG0187 for 48 h with or without 5 ng/ml TGF-β for 6 h and subjected to quantitative PCR analysis. mRNA levels of PAI-1, CTGF, and IL-11 were normalized to GAPDH expression. (C) MDA-MB-231 cells were pretreated for 6 h with 1 ng/ml GLPG0187 or vehicle, then injected into the blood circulation of zebrafish 48-h post fertilization and followed with or without 0.5 ng/ml GLPG0187 treatment, as indicated. Confocal images of overview zebrafish were made 5 days post implantion (dpi). Arrows indicate invasive tumor cells, scale bar: 500 μm. (D) Percentage of embryos displaying invasion and metastasis at 1, 3, and 5 dpi is shown; vehicle versus GLPG0187 P <0.001. (E) Representative images of the zebrafish described in panel D. Experimental micrometastasis was detected in the posterior tail fin at 5 dpi. Arrows indicate invasive tumor cells, scale bar: 100 μm. less

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A functional genome-wide in vivo screen identifies new regulators of signalling pathways during early Xenopus embryogenesis.

In PLoS One on 19 November 2013 by Zhang, S., Li, J., et al.

Fig.6.D
Fig.6.D showing Western Blotting from the publication: A functional genome-wide in vivo screen identifies new regulators of signalling pathways during early Xenopus embryogenesis.
  • WB
Results of the screen.The 20 active clones identified during the screen were individually injected and analysed by Western blot to demonstrate their abilities to modulate the activities of different signalling pathways as shown in Table 3. Embryos were collected at the indicated stages, processed... more
Results of the screen.The 20 active clones identified during the screen were individually injected and analysed by Western blot to demonstrate their abilities to modulate the activities of different signalling pathways as shown in Table 3. Embryos were collected at the indicated stages, processed, and analysed by Western blot to assess the activities of Erk (pErk, panel A), Akt (pAkt, panel B), BMP (pSmad1, panel C), and TGFβ/Nodal (pSmad2, panel D). Control (ctrl) denotes uninjected embryos. Anti-Erk (Erk), anti-Akt (Akt), anti-Smad2 (Smad2) and anti-α-Tubulin (α-Tubulin) were used as loading controls. less

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A functional genome-wide in vivo screen identifies new regulators of signalling pathways during early Xenopus embryogenesis.

In PLoS One on 19 November 2013 by Zhang, S., Li, J., et al.

Fig.4.C
Fig.4.C showing Western Blotting from the publication: A functional genome-wide in vivo screen identifies new regulators of signalling pathways during early Xenopus embryogenesis.
  • WB
Proof of principle of the screen.(A) 12 pools, each one with one clone of known activity, were selected from the full-length EST library and injected into embryos as described. Protein extracts from collected embryos were subjected to Western blot using indicated antibodies to observe phosphoryla... more
Proof of principle of the screen.(A) 12 pools, each one with one clone of known activity, were selected from the full-length EST library and injected into embryos as described. Protein extracts from collected embryos were subjected to Western blot using indicated antibodies to observe phosphorylation changes of specific signalling molecules at blastula and gastrula stages. Note the reduction of phospho-Smad2 activity at gastrula stage on pool 2, and reduction of phospho-Smad1/5/8 activity on pool 11. (B) De-convolution of pool 2. cerberus is identified as a negative regulator of Smad2 (pSmad2, lower panel) but not of Smad1 (pSmad1, upper panel) phosphorylation at gastrula stage. (C) De-convolution of pool 11. wnt8a is identified as a negative regulator of Smad1/5/8 phosphorylation (pSmad1, middle panel) and activator of Wnt signalling (pLRP6, upper panel) at gastrula stage. UI: uninjected. less

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A functional genome-wide in vivo screen identifies new regulators of signalling pathways during early Xenopus embryogenesis.

In PLoS One on 19 November 2013 by Zhang, S., Li, J., et al.

Fig.4.A
Fig.4.A showing Western Blotting from the publication: A functional genome-wide in vivo screen identifies new regulators of signalling pathways during early Xenopus embryogenesis.
  • WB
Proof of principle of the screen.(A) 12 pools, each one with one clone of known activity, were selected from the full-length EST library and injected into embryos as described. Protein extracts from collected embryos were subjected to Western blot using indicated antibodies to observe phosphoryla... more
Proof of principle of the screen.(A) 12 pools, each one with one clone of known activity, were selected from the full-length EST library and injected into embryos as described. Protein extracts from collected embryos were subjected to Western blot using indicated antibodies to observe phosphorylation changes of specific signalling molecules at blastula and gastrula stages. Note the reduction of phospho-Smad2 activity at gastrula stage on pool 2, and reduction of phospho-Smad1/5/8 activity on pool 11. (B) De-convolution of pool 2. cerberus is identified as a negative regulator of Smad2 (pSmad2, lower panel) but not of Smad1 (pSmad1, upper panel) phosphorylation at gastrula stage. (C) De-convolution of pool 11. wnt8a is identified as a negative regulator of Smad1/5/8 phosphorylation (pSmad1, middle panel) and activator of Wnt signalling (pLRP6, upper panel) at gastrula stage. UI: uninjected. less

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A functional genome-wide in vivo screen identifies new regulators of signalling pathways during early Xenopus embryogenesis.

In PLoS One on 19 November 2013 by Zhang, S., Li, J., et al.

Fig.4.B
Fig.4.B showing Western Blotting from the publication: A functional genome-wide in vivo screen identifies new regulators of signalling pathways during early Xenopus embryogenesis.
  • WB
Proof of principle of the screen.(A) 12 pools, each one with one clone of known activity, were selected from the full-length EST library and injected into embryos as described. Protein extracts from collected embryos were subjected to Western blot using indicated antibodies to observe phosphoryla... more
Proof of principle of the screen.(A) 12 pools, each one with one clone of known activity, were selected from the full-length EST library and injected into embryos as described. Protein extracts from collected embryos were subjected to Western blot using indicated antibodies to observe phosphorylation changes of specific signalling molecules at blastula and gastrula stages. Note the reduction of phospho-Smad2 activity at gastrula stage on pool 2, and reduction of phospho-Smad1/5/8 activity on pool 11. (B) De-convolution of pool 2. cerberus is identified as a negative regulator of Smad2 (pSmad2, lower panel) but not of Smad1 (pSmad1, upper panel) phosphorylation at gastrula stage. (C) De-convolution of pool 11. wnt8a is identified as a negative regulator of Smad1/5/8 phosphorylation (pSmad1, middle panel) and activator of Wnt signalling (pLRP6, upper panel) at gastrula stage. UI: uninjected. less

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A functional genome-wide in vivo screen identifies new regulators of signalling pathways during early Xenopus embryogenesis.

In PLoS One on 19 November 2013 by Zhang, S., Li, J., et al.

Fig.1.B
Fig.1.B showing Western Blotting in a Xenopus laevis (African clawed frog) sample from the publication: A functional genome-wide in vivo screen identifies new regulators of signalling pathways during early Xenopus embryogenesis.
  • WB
  • Xenopus laevis (African clawed frog)
Characterisation of phospho-specific antibodies.(A) Characterisation of anti-phospho-Akt (pAkt) and anti-phospho-Erk (pErk) antibodies, the PI3K/Akt inhibitor LY294002 and FGF inhibitor SU5402 were used to inhibit Akt and Erk phosphorylation in gastrula embryos, respectively. 1% DMSO was used to ... more
Characterisation of phospho-specific antibodies.(A) Characterisation of anti-phospho-Akt (pAkt) and anti-phospho-Erk (pErk) antibodies, the PI3K/Akt inhibitor LY294002 and FGF inhibitor SU5402 were used to inhibit Akt and Erk phosphorylation in gastrula embryos, respectively. 1% DMSO was used to exclude any possible interference from the inhibitor solvent. Anti-Erk (Erk), anti-Akt (Akt) and anti-α-tubulin (α-tubulin) were used as loading controls. (B) Characterisation of anti-phospho-Smad1/5/8 (pSmad1) and anti-phospho-Smad2 (pSmad2) antibodies. The TGF-βRI inhibitor SB505124 was used to inhibit Smad2 phosphorylation in gastrula embryos; injection of wnt8a mRNA was used to inhibit bmp4 expression, thus preventing Smad1/5/8 phosphorylation. All inhibitors have been added at stage 6. 1% DMSO was used to exclude any possible interference from the inhibitor solvent. Smad2 and α-tubulin serves as internal controls to ensure equal loading in all lanes. less

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