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Fig.1.B showing Western Blotting from the publication: RNA-binding is an ancient trait of the Annexin family.

Fig.1.A showing Western Blotting from the publication: RNA-binding is an ancient trait of the Annexin family.

Fig.3.C showing Western Blotting in a Homo sapiens (Human) sample from the publication: Exophagy of annexin A2 via RAB11, RAB8A and RAB27A in IFN-γ-stimulated lung epithelial cells.

Fig.1.D showing Western Blotting in a Homo sapiens (Human) sample from the publication: Exophagy of annexin A2 via RAB11, RAB8A and RAB27A in IFN-γ-stimulated lung epithelial cells.

Fig.5.C showing Western Blotting in a Homo sapiens (Human) sample from the publication: Exophagy of annexin A2 via RAB11, RAB8A and RAB27A in IFN-γ-stimulated lung epithelial cells.

Fig.2.E showing Western Blotting in a Homo sapiens (Human) sample from the publication: Exophagy of annexin A2 via RAB11, RAB8A and RAB27A in IFN-γ-stimulated lung epithelial cells.

Fig.4.C showing Western Blotting in a Homo sapiens (Human) sample from the publication: Exophagy of annexin A2 via RAB11, RAB8A and RAB27A in IFN-γ-stimulated lung epithelial cells.

Fig.4.B showing Western Blotting in a Rattus norvegicus (Rat) sample from the publication: Post-translational modifications of Annexin A2 are linked to its association with perinuclear nonpolysomal mRNP complexes.

Fig.1.A showing Western Blotting in a Rattus norvegicus (Rat) sample from the publication: Post-translational modifications of Annexin A2 are linked to its association with perinuclear nonpolysomal mRNP complexes.

Fig.4.D showing Western Blotting in a Rattus norvegicus (Rat) sample from the publication: Post-translational modifications of Annexin A2 are linked to its association with perinuclear nonpolysomal mRNP complexes.

Fig.2.B showing Western Blotting in a Rattus norvegicus (Rat) sample from the publication: Post-translational modifications of Annexin A2 are linked to its association with perinuclear nonpolysomal mRNP complexes.

Fig.2.A showing Western Blotting in a Rattus norvegicus (Rat) sample from the publication: Post-translational modifications of Annexin A2 are linked to its association with perinuclear nonpolysomal mRNP complexes.

Fig.5.A showing Western Blotting in a Rattus norvegicus (Rat) sample from the publication: Reactive oxygen species exert opposite effects on Tyr23 phosphorylation of the nuclear and cortical pools of annexin A2.

Fig.5.B showing Immunoprecipitation in a Rattus norvegicus (Rat) sample from the publication: Reactive oxygen species exert opposite effects on Tyr23 phosphorylation of the nuclear and cortical pools of annexin A2.

Fig.1.A showing Western Blotting in a Homo sapiens (Human) sample from the publication: Genotoxic agents promote the nuclear accumulation of annexin A2: role of annexin A2 in mitigating DNA damage.

Fig.1.G showing Western Blotting in a Homo sapiens (Human) sample from the publication: Genotoxic agents promote the nuclear accumulation of annexin A2: role of annexin A2 in mitigating DNA damage.

Fig.1.E showing Western Blotting in a Homo sapiens (Human) sample from the publication: Genotoxic agents promote the nuclear accumulation of annexin A2: role of annexin A2 in mitigating DNA damage.

Fig.1.F showing Western Blotting in a Homo sapiens (Human) sample from the publication: Genotoxic agents promote the nuclear accumulation of annexin A2: role of annexin A2 in mitigating DNA damage.

Fig.1.D showing Western Blotting in a Homo sapiens (Human) sample from the publication: Genotoxic agents promote the nuclear accumulation of annexin A2: role of annexin A2 in mitigating DNA damage.

Fig.1.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: Genotoxic agents promote the nuclear accumulation of annexin A2: role of annexin A2 in mitigating DNA damage.

Fig.5.A showing Western Blotting in a Homo sapiens (Human) sample from the publication: Genotoxic agents promote the nuclear accumulation of annexin A2: role of annexin A2 in mitigating DNA damage.

Fig.2.A showing Western Blotting in a Homo sapiens (Human) sample from the publication: Genotoxic agents promote the nuclear accumulation of annexin A2: role of annexin A2 in mitigating DNA damage.

Fig.4.A showing Western Blotting in a Homo sapiens (Human) sample from the publication: Genotoxic agents promote the nuclear accumulation of annexin A2: role of annexin A2 in mitigating DNA damage.

Fig.4.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: Genotoxic agents promote the nuclear accumulation of annexin A2: role of annexin A2 in mitigating DNA damage.

Fig.4.C showing Western Blotting in a Homo sapiens (Human) sample from the publication: Genotoxic agents promote the nuclear accumulation of annexin A2: role of annexin A2 in mitigating DNA damage.

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Annexin A2 promotes proliferative vitreoretinopathy in response to a macrophage inflammatory signal in mice.

In Nature Communications on 9 October 2024 by Luo, M., Almeida, D., et al.

Proliferative vitreoretinopathy is a vision-threatening response to penetrating ocular injury, for which there is no satisfactory treatment. In this disorder, retinal pigment epithelial cells, abandon their attachment to Bruch's membrane on the scleral side of the retina, transform into motile fibroblast-like cells, and migrate through the retinal wound to the vitreal surface of the retina, where they secrete membrane-forming proteins. Annexin A2 is a calcium-regulated protein that, in complex with S100A10, assembles plasmin-forming proteins at cell surfaces. Here, we show that, in proliferative vitreoretinopathy, recruitment of macrophages and directed migration of retinal pigment epithelial cells are annexin A2-dependent, and stimulated by macrophage inflammatory protein-1α/β. These factors induce translocation of annexin A2 to the cell surface, thus enabling retinal pigment epithelial cell migration following injury; our studies reveal further that treatment of mice with intraocular antibody to either annexin A2 or macrophage inflammatory protein dampens the development of proliferative vitreoretinopathy in mice.
© 2024. The Author(s).

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Disabled-2: a protein up-regulated by high molecular weight hyaluronan has both tumor promoting and tumor suppressor roles in ovarian cancer.

In Cellular and Molecular Life Sciences : CMLS on 10 October 2023 by Price, Z. K., Lokman, N. A., et al.

Although the pro-tumorigenic functions of hyaluronan (HA) are well documented there is limited information on the effects and targets of different molecular weight HA. Here, we investigated the effects of 27 kDa, 183 kDa and 1000 kDa HA on ES-2 ovarian cancer cells overexpressing the stem cell associated protein, Notch3. 1000 kDA HA promoted spheroid formation in ES-2 cells mixed with ES-2 overexpressing Notch3 (1:3). We report disabled-2 (DAB2) as a novel protein regulated by 1000 kDa HA and further investigated its role in ovarian cancer. DAB2 was downregulated in ovarian cancer compared to normal tissues but increased in metastatic ovarian tumors compared to primary tumors. High DAB2 expression was associated with poor patient outcome and positively correlated with HA synthesis enzyme HAS2, HA receptor CD44 and EMT and macrophage markers. Stromal DAB2 immunostaining was significantly increased in matched ovarian cancer tissues at relapse compared to diagnosis and associated with reduced survival. The proportion of DAB2 positive macrophages was significantly increased in metastatic ovarian cancer tissues compared to primary cancers. However, DAB2 overexpression significantly reduced invasion by both A2780 and OVCAR3 cells in vivo. Our research identifies a novel relationship between HA signalling, Notch3 and DAB2. We highlight a complex relationship of both pro-tumorigenic and tumor suppressive functions of DAB2 in ovarian cancer. Our findings highlight that DAB2 has a direct tumor suppressive role on ovarian cancer cells. The pro-tumorigenic role of DAB2 may be mediated by tumour associated macrophages and requires further investigation.
© 2023. The Author(s).

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Disabled-2: a protein up-regulated by high molecular weight hyaluronan has both tumor promoting and tumor suppressor roles in ovarian cancer

Preprint on Research Square on 12 July 2023 by Price, Z. K., Lokman, N. A., et al.

Although the pro-tumorigenic functions of hyaluronan (HA) are well documented there is limited information on the effects and targets of different molecular weight HA. Here, we investigated the effects of 27kDa, 183kDa and 1000kDa HA on ES2 ovarian cancer cells overexpressing the stem cell associated protein, Notch3. 1000kDA HA promoted spheroid formation in ES2 cells mixed with ES-2 overexpressing Notch3 (1:3). We report disabled-2 (DAB2) as a novel protein regulated by high molecular weight HA and further investigated its role in ovarian cancer. DAB2 was downregulated in ovarian cancer compared to normal tissues but increased in metastatic ovarian tumors compared to primary tumors. High DAB2 expression was associated with poor patient outcome and positively correlated with HA synthesis enzyme HAS2 , HA receptor, CD44 and EMT and macrophage markers. Stromal DAB2 immunostaining was significantly increased in matched ovarian cancer tissues at relapse compared to diagnosis and associated with reduced survival. However, DAB2 overexpression significantly reduced invasion by both A2780 and OVCAR3 cells in vivo . Our research identifies a novel relationship between HA and DAB2. Furthermore, we highlight a complex relationship of both pro-tumorigenic and tumor suppressive functions of DAB2 in ovarian cancer. Further research should explore the pro-tumorigenic role of DAB2 within the tumor microenvironment of ovarian cancer.

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RNA-binding is an ancient trait of the Annexin family.

In Frontiers in Cell and Developmental Biology on 3 July 2023 by Patil, S. S., Panchal, V., et al.

Introduction: The regulation of intracellular functions in mammalian cells involves close coordination of cellular processes. During recent years it has become evident that the sorting, trafficking and distribution of transport vesicles and mRNA granules/complexes are closely coordinated to ensure effective simultaneous handling of all components required for a specific function, thereby minimizing the use of cellular energy. Identification of proteins acting at the crossroads of such coordinated transport events will ultimately provide mechanistic details of the processes. Annexins are multifunctional proteins involved in a variety of cellular processes associated with Ca2+-regulation and lipid binding, linked to the operation of both the endocytic and exocytic pathways. Furthermore, certain Annexins have been implicated in the regulation of mRNA transport and translation. Since Annexin A2 binds specific mRNAs via its core structure and is also present in mRNP complexes, we speculated whether direct association with RNA could be a common property of the mammalian Annexin family sharing a highly similar core structure. Methods and results: Therefore, we performed spot blot and UV-crosslinking experiments to assess the mRNA binding abilities of the different Annexins, using annexin A2 and c-myc 3'UTRs as well as c-myc 5'UTR as baits. We supplemented the data with immunoblot detection of selected Annexins in mRNP complexes derived from the neuroendocrine rat PC12 cells. Furthermore, biolayer interferometry was used to determine the KD of selected Annexin-RNA interactions, which indicated distinct affinities. Amongst these Annexins, Annexin A13 and the core structures of Annexin A7, Annexin A11 bind c-myc 3'UTR with KDs in the nanomolar range. Of the selected Annexins, only Annexin A2 binds the c-myc 5'UTR indicating some selectivity. Discussion: The oldest members of the mammalian Annexin family share the ability to associate with RNA, suggesting that RNA-binding is an ancient trait of this protein family. Thus, the combined RNA- and lipid-binding properties of the Annexins make them attractive candidates to participate in coordinated long-distance transport of membrane vesicles and mRNAs regulated by Ca2+. The present screening results can thus pave the way for studies of the multifunctional Annexins in a novel cellular context.
Copyright © 2023 Patil, Panchal, Røstbø, Romanyuk, Hollås, Brenk, Grindheim and Vedeler.

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Disabled-2: a protein up-regulated by high molecular weight hyaluronan has both tumor suppressor and tumor promoting roles in ovarian cancer

Preprint on Research Square on 26 June 2023 by Price, Z. K., Lokman, N. A., et al.

Objective: Although the pro-tumorigenic functions of hyaluronan (HA) are well documented in ovarian cancer, there is limited information on the effects of different molecular weight HA. The aim of this study was to analyse the effects of different molecular weight HA on ovarian cancer cells overexpressing Notch3 intracellular domain (NICD3, stem cell associated protein). Methods: : Mass spectrometry analysis of spheroids from ES-2 cells overexpressing NICD3 (ES-2-Rv-NICD3) with wild type ES-2 (ES-2:ES-2-Rv-NICD3, 1:3) treated with 27kDa, 183kDa or 1000kDa HA identified a novel protein regulated by high molecular weight HA (HMW-HA), disabled-2 (DAB2). Correlations between DAB2 and patient prognosis and pro-tumorigenic signatures were assessed in online databases. DAB2 was assessed by immunohistochemistry in a tissue microarray cohort of high grade serous ovarian carcinoma (HGSOC) and matching tissues following relapse. Gain-of-function lentiviral methods were employed in A2780 and OVCAR3 ovarian cancer cells to determine the effect of DAB2 on cell survival, spheroid formation, gene expression, cell motility and invasion in vitro and in vivo using the chick chorioallantoic membrane (CAM) assay. Results: : HMW-HA (1000kDa) enhanced spheroid formation of ES-2:ES-2-Rv-NICD3 cells. Mass spectrometry identified DAB2 was upregulated 5.2 fold in HMW-HA treated ES-2:ES-2-Rv-NICD3 spheroids. Online database analysis showed DAB2 was downregulated in ovarian cancer compared to normal ovarian tissue but increased in metastatic compared to primary ovarian tumors. High DAB2 expression was associated with poor patient outcome and positively correlated with EMT markers . Stromal DAB2 immunostaining was significantly increased in matched tissues at relapse compared to diagnosis and associated with reduced survival. Furthermore, DAB2 protein co-localised with macrophage marker (CD68) in HGSOC tissues. In OVCAR3 but not A2780 cells, DAB2 overexpression enhanced carboplatin resistance and reduced cell motility and invasion in vitro . DAB2 overexpression reduced OVCAR3 and A2780 cell survival and in vivo invasion in the CAM assay. Conclusions: : Our findings highlight that DAB2 has both tumor suppressive and pro-tumorigenic functions in ovarian cancer

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RNA-binding is an ancient trait of the Annexin family.

In Front Cell Dev Biol on 3 July 2023 by Patil, S. S., Panchal, V., et al.

Fig.1.B

Fig.1.B showing Western Blotting from the publication: RNA-binding is an ancient trait of the Annexin family.

AnxA1, AnxA2, AnxA4, AnxA5, AnxA6, AnxA7, AnxA10, AnxA11 and AnxA13 present in the cytoskeleton fraction (Panel A) are associated with non-polysomal mRNP complexes (Panel B) of PC12 cells. Panel (A) 30 µg of the cytoskeletal fraction (lane 1) and cytoskeleton-bound polysomes (lane 2) were separat... more

AnxA1, AnxA2, AnxA4, AnxA5, AnxA6, AnxA7, AnxA10, AnxA11 and AnxA13 present in the cytoskeleton fraction (Panel A) are associated with non-polysomal mRNP complexes (Panel B) of PC12 cells. Panel (A) 30 µg of the cytoskeletal fraction (lane 1) and cytoskeleton-bound polysomes (lane 2) were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. Panel (B) samples prepared from oligo (dT)-bound mRNP complexes from the cytoskeletal fraction [supernatant after centrifugation for 2 h 100,000 g above a 1 M (35%) sucrose cushion] (lanes 3 and 4), without (lane 3) or with RNase (lane 4) treatment, as indicated above the Western blots, were subjected to similar analysis. The blots were probed with antibodies against the different Anxs and against PABP1 as a marker for poly(A)-containing mRNAs, as indicated. Antibodies against the ribosomal subunit S6 were used to inform of the distribution of ribosomes. In addition, the blots were probed with antibodies against early endosomes (EEA1), late endosomes (Rab7) and recycling endosomes (Rab11). SPC25 and LAMP1 were not detectable in any of the fractions (results not shown). Visualization of the immunoreactive protein bands was performed using the ChemiDocTM XRS+ molecular imager after incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies and enhanced chemiluminescence (ECL)-reagent. The blots shown are representative for results from three experiments. less

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RNA-binding is an ancient trait of the Annexin family.

In Front Cell Dev Biol on 3 July 2023 by Patil, S. S., Panchal, V., et al.

Fig.1.A

Fig.1.A showing Western Blotting from the publication: RNA-binding is an ancient trait of the Annexin family.

AnxA1, AnxA2, AnxA4, AnxA5, AnxA6, AnxA7, AnxA10, AnxA11 and AnxA13 present in the cytoskeleton fraction (Panel A) are associated with non-polysomal mRNP complexes (Panel B) of PC12 cells. Panel (A) 30 µg of the cytoskeletal fraction (lane 1) and cytoskeleton-bound polysomes (lane 2) were separat... more

AnxA1, AnxA2, AnxA4, AnxA5, AnxA6, AnxA7, AnxA10, AnxA11 and AnxA13 present in the cytoskeleton fraction (Panel A) are associated with non-polysomal mRNP complexes (Panel B) of PC12 cells. Panel (A) 30 µg of the cytoskeletal fraction (lane 1) and cytoskeleton-bound polysomes (lane 2) were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. Panel (B) samples prepared from oligo (dT)-bound mRNP complexes from the cytoskeletal fraction [supernatant after centrifugation for 2 h 100,000 g above a 1 M (35%) sucrose cushion] (lanes 3 and 4), without (lane 3) or with RNase (lane 4) treatment, as indicated above the Western blots, were subjected to similar analysis. The blots were probed with antibodies against the different Anxs and against PABP1 as a marker for poly(A)-containing mRNAs, as indicated. Antibodies against the ribosomal subunit S6 were used to inform of the distribution of ribosomes. In addition, the blots were probed with antibodies against early endosomes (EEA1), late endosomes (Rab7) and recycling endosomes (Rab11). SPC25 and LAMP1 were not detectable in any of the fractions (results not shown). Visualization of the immunoreactive protein bands was performed using the ChemiDocTM XRS+ molecular imager after incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies and enhanced chemiluminescence (ECL)-reagent. The blots shown are representative for results from three experiments. less

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Exophagy of annexin A2 via RAB11, RAB8A and RAB27A in IFN-γ-stimulated lung epithelial cells.

In Sci Rep on 18 July 2017 by Chen, Y. D., Fang, Y. T., et al.

Fig.1.D

Fig.1.D showing Western Blotting in a Homo sapiens (Human) sample from the publication: Exophagy of annexin A2 via RAB11, RAB8A and RAB27A in IFN-γ-stimulated lung epithelial cells.

IFN-γ-induced autophagy is required for extracellular secretion of ANXA2 in human lung epithelial cells. (a) A549 cells were treated with or without 500 U/ml IFN-γ for different time periods. After fixation and permeabilization, cells were stained with anti-LC3 (green) and DAPI (blue) to detect L... more

IFN-γ-induced autophagy is required for extracellular secretion of ANXA2 in human lung epithelial cells. (a) A549 cells were treated with or without 500 U/ml IFN-γ for different time periods. After fixation and permeabilization, cells were stained with anti-LC3 (green) and DAPI (blue) to detect LC3 puncta and nuclei, respectively, and observed by confocal microscopy. Scale bar: 10 μm (2 μm in insets). (b) The quantification of LC3 puncta per cell is shown. Data are represented as mean ± SD. ***P < 0.001. At least 30 cells were counted in each treatment group. (c) Cells were cultured with 500 U/ml IFN-γ for the indicated times. Cells were harvested and lysed after stimulation. The expression of SQSTM1/p62, LC3, cathepsin S and α-tubulin were analyzed by western blotting. The densitometric ratio of LC3-II and α-tubulin is shown. kD, molecular weight as kDa. (d) Cells were pretreated in the presence or absence of 3-MA for 1 h and then stimulated with or without 500 U/ml IFN-γ. After 48 h, ANXA2 and α-tubulin from cultured supernatant and lysate were analyzed by western blotting. (e) Cells were transfected with shRNA specifically targeting luciferase or ATG5. Cells were cultured with or without 500 U/ml IFN-γ for 48 h. ANXA2, calnexin and α-tubulin from cultured supernatant and cell lysate were analyzed by western blotting. ATG5 knockdown efficiency was detected by western blotting. less

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Post-translational modifications of Annexin A2 are linked to its association with perinuclear nonpolysomal mRNP complexes.

In FEBS Open Bio on 1 February 2017 by Aukrust, I., Rosenberg, L. A., et al.

Fig.4.B

Fig.4.B showing Western Blotting in a Rattus norvegicus (Rat) sample from the publication: Post-translational modifications of Annexin A2 are linked to its association with perinuclear nonpolysomal mRNP complexes.

pSer25AnxA2 and ubiquitinated high‐molecular‐mass forms of AnxA2 associate with translationally inactive mRNP complexes. (A, B) High‐molecular‐mass forms of pSer25AnxA2 is present in oligo(dT)‐purified nonpolysomal mRNP complexes in PC12 cells. (A) Schematic representation of the method used in (... more

pSer25AnxA2 and ubiquitinated high‐molecular‐mass forms of AnxA2 associate with translationally inactive mRNP complexes. (A, B) High‐molecular‐mass forms of pSer25AnxA2 is present in oligo(dT)‐purified nonpolysomal mRNP complexes in PC12 cells. (A) Schematic representation of the method used in (B) with reference to the individual lanes in (B). (B) Samples (100 μg of protein) were prepared from the following fractions: nucleus (lane 1), supernatant (lane 2) and polysome‐containing pellet (lane 5; derived from the nuclear fraction after centrifugation for 2 h 100 000 g through a 1 m sucrose cushion), non‐oligo(dT)‐bound supernatant (lane 3), oligo(dT)‐bound supernatant (lane 4), and oligo(dT)‐bound pellet (lane 6), as indicated above the western blot. The proteins were separated by 10% SDS/PAGE and subjected to western blot analysis. The blots were probed with antibodies against pSer25AnxA2, total AnxA2, tubulin, SPC, S6 kinase and the ribosomal protein S6, as indicated. Detection of the resulting protein bands was performed by the ChemiDoc™ XRS+ molecular imager after incubation with HRP‐conjugated secondary antibodies and ECL reagent. The arrowheads to the left indicate the protein molecular mass standards. (C, D) High‐molecular‐mass forms of AnxA2 in mRNP complexes affinity‐purified via binding to anxA2 mRNA represent ubiquitinated forms of the protein. (C) Schematic overview of the method used in (D) with reference to the individual lanes in (D). Proteins (100 μg) present in the total cytoskeletal fraction derived from NGF‐stimulated PC12 cells (lanes 1 and 5), the unbound fraction (lanes 2 and 6) and AnxA2 IP proteins from the affinity‐purified mRNP complexes derived from the cytoskeletal fraction (lanes 3 and 7), were subjected to 10% SDS/PAGE and immunoblot analysis using monoclonal antibodies against AnxA2 (lanes 1–4) or Ub (lane 5–7). The bands representing ubiquitinated AnxA2 are indicated by the upper bracket to the right. Lane 4 represents a negative control, showing the binding of cytoskeleton‐associated proteins to anxA2 mRNA coupled to oligo(dT) magnetic beads in the presence of RNase, followed by IP using monoclonal AnxA2 antibodies. The molecular mass markers are indicated to the left and the IgG heavy (Hc; arrowhead) and light (Lc; lower bracket) chains to the right. less

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Post-translational modifications of Annexin A2 are linked to its association with perinuclear nonpolysomal mRNP complexes.

In FEBS Open Bio on 1 February 2017 by Aukrust, I., Rosenberg, L. A., et al.

Fig.1.A

Fig.1.A showing Western Blotting in a Rattus norvegicus (Rat) sample from the publication: Post-translational modifications of Annexin A2 are linked to its association with perinuclear nonpolysomal mRNP complexes.

Detection of pSer25AnxA2 in subcellular fractions derived from PC12 cells. Proteins (100 μg) from the cytoplasm (lane 1), the cytoplasm devoid of mitochondria (‐mit; lane 2), the cytosol (lane 3), the cytoskeleton (lane 4), ER (lane 5), mitochondria (lane 6), the nuclear fraction (lane 7) and EGT... more

Detection of pSer25AnxA2 in subcellular fractions derived from PC12 cells. Proteins (100 μg) from the cytoplasm (lane 1), the cytoplasm devoid of mitochondria (‐mit; lane 2), the cytosol (lane 3), the cytoskeleton (lane 4), ER (lane 5), mitochondria (lane 6), the nuclear fraction (lane 7) and EGTA‐released ECM (lane 8) were separated by 10% SDS/PAGE and subjected to western blot analysis. The blots were probed with antibodies against pSer25AnxA2 and total AnxA2, as indicated. Antibodies against compartmental markers, namely the cytoplasm (tubulin; 55 kDa), ER (SPC; 25 kDa), nucleus (fibrillarin; 35 kDa) and mitochondria (complex II; 70 kDa) were also employed as indicated. The blot probed against pSer25AnxA2 was reprobed against fibrillarin, while the blot probed against AnxA2 was reprobed against SPC. Only 25 μg of protein from the mitochondrial fraction was used for western blot analysis of tubulin and complex II on two different membranes. Detection of the immunoreactive protein bands was performed using the ChemiDoc™ XRS+ molecular imager after incubation with HRP‐conjugated secondary antibodies and enhanced chemiluminescence (ECL) reagent. The methods (A–D) used to generate the different fractions are indicated above the western blots and described in the Methods section. The arrowheads to the left indicate the protein molecular mass standards. less

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Post-translational modifications of Annexin A2 are linked to its association with perinuclear nonpolysomal mRNP complexes.

In FEBS Open Bio on 1 February 2017 by Aukrust, I., Rosenberg, L. A., et al.

Fig.4.D

Fig.4.D showing Western Blotting in a Rattus norvegicus (Rat) sample from the publication: Post-translational modifications of Annexin A2 are linked to its association with perinuclear nonpolysomal mRNP complexes.

pSer25AnxA2 and ubiquitinated high‐molecular‐mass forms of AnxA2 associate with translationally inactive mRNP complexes. (A, B) High‐molecular‐mass forms of pSer25AnxA2 is present in oligo(dT)‐purified nonpolysomal mRNP complexes in PC12 cells. (A) Schematic representation of the method used in (... more

pSer25AnxA2 and ubiquitinated high‐molecular‐mass forms of AnxA2 associate with translationally inactive mRNP complexes. (A, B) High‐molecular‐mass forms of pSer25AnxA2 is present in oligo(dT)‐purified nonpolysomal mRNP complexes in PC12 cells. (A) Schematic representation of the method used in (B) with reference to the individual lanes in (B). (B) Samples (100 μg of protein) were prepared from the following fractions: nucleus (lane 1), supernatant (lane 2) and polysome‐containing pellet (lane 5; derived from the nuclear fraction after centrifugation for 2 h 100 000 g through a 1 m sucrose cushion), non‐oligo(dT)‐bound supernatant (lane 3), oligo(dT)‐bound supernatant (lane 4), and oligo(dT)‐bound pellet (lane 6), as indicated above the western blot. The proteins were separated by 10% SDS/PAGE and subjected to western blot analysis. The blots were probed with antibodies against pSer25AnxA2, total AnxA2, tubulin, SPC, S6 kinase and the ribosomal protein S6, as indicated. Detection of the resulting protein bands was performed by the ChemiDoc™ XRS+ molecular imager after incubation with HRP‐conjugated secondary antibodies and ECL reagent. The arrowheads to the left indicate the protein molecular mass standards. (C, D) High‐molecular‐mass forms of AnxA2 in mRNP complexes affinity‐purified via binding to anxA2 mRNA represent ubiquitinated forms of the protein. (C) Schematic overview of the method used in (D) with reference to the individual lanes in (D). Proteins (100 μg) present in the total cytoskeletal fraction derived from NGF‐stimulated PC12 cells (lanes 1 and 5), the unbound fraction (lanes 2 and 6) and AnxA2 IP proteins from the affinity‐purified mRNP complexes derived from the cytoskeletal fraction (lanes 3 and 7), were subjected to 10% SDS/PAGE and immunoblot analysis using monoclonal antibodies against AnxA2 (lanes 1–4) or Ub (lane 5–7). The bands representing ubiquitinated AnxA2 are indicated by the upper bracket to the right. Lane 4 represents a negative control, showing the binding of cytoskeleton‐associated proteins to anxA2 mRNA coupled to oligo(dT) magnetic beads in the presence of RNase, followed by IP using monoclonal AnxA2 antibodies. The molecular mass markers are indicated to the left and the IgG heavy (Hc; arrowhead) and light (Lc; lower bracket) chains to the right. less

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Post-translational modifications of Annexin A2 are linked to its association with perinuclear nonpolysomal mRNP complexes.

In FEBS Open Bio on 1 February 2017 by Aukrust, I., Rosenberg, L. A., et al.

Fig.2.B

Fig.2.B showing Western Blotting in a Rattus norvegicus (Rat) sample from the publication: Post-translational modifications of Annexin A2 are linked to its association with perinuclear nonpolysomal mRNP complexes.

Distribution of pSer25AnxA2 in nuclear subfractions of PC12 cells (A). Proteins (100 μg) from the nucleoplasm (lane 1) and NE (lane 2) fractions were separated by 10% SDS/PAGE and subjected to western blot analysis. The blot was probed with antibodies against pSer25AnxA2, total AnxA2, tubulin, SP... more

Distribution of pSer25AnxA2 in nuclear subfractions of PC12 cells (A). Proteins (100 μg) from the nucleoplasm (lane 1) and NE (lane 2) fractions were separated by 10% SDS/PAGE and subjected to western blot analysis. The blot was probed with antibodies against pSer25AnxA2, total AnxA2, tubulin, SPC and fibrillarin, as indicated. myr‐ψ‐PKC inhibits Ser25 phosphorylation of AnxA2 and shifts the localisation of AnxA2 from the perinuclear to the cortical region of PC12 cells (B, C). Proteins (100 μg) from the cytoplasmic (lanes 1 and 2) and nuclear (lanes 3 and 4) fractions prepared from myr‐ψ‐PKC‐treated (+; lanes 2 and 4) and control (−; lanes 1 and 3) cells were separated by 10% SDS/PAGE and subjected to western blot analysis (B). The blots were probed with antibodies against pSer25AnxA2, total AnxA2, tubulin and GAPDH, as indicated. Detection of the resulting protein bands (A, B) was performed using the ChemiDoc™ XRS+ molecular imager after incubation with HRP‐conjugated secondary antibodies and ECL reagent. The arrows to the left indicate the protein molecular mass standards (nd, not determined). Immunofluorescence staining of control and myr‐ψ‐PKC‐treated cells, as indicated, was carried out using rabbit polyclonal antibodies against AnxA2 (green) (C). DNA staining with DAPI (blue fluorescence) was included to visualise the nucleus. Scale bar is 10 μm. The insets show higher magnification of the indicated areas in the images shown to the right in Panel (C). less

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Post-translational modifications of Annexin A2 are linked to its association with perinuclear nonpolysomal mRNP complexes.

In FEBS Open Bio on 1 February 2017 by Aukrust, I., Rosenberg, L. A., et al.

Fig.2.A

Fig.2.A showing Western Blotting in a Rattus norvegicus (Rat) sample from the publication: Post-translational modifications of Annexin A2 are linked to its association with perinuclear nonpolysomal mRNP complexes.

Distribution of pSer25AnxA2 in nuclear subfractions of PC12 cells (A). Proteins (100 μg) from the nucleoplasm (lane 1) and NE (lane 2) fractions were separated by 10% SDS/PAGE and subjected to western blot analysis. The blot was probed with antibodies against pSer25AnxA2, total AnxA2, tubulin, SP... more

Distribution of pSer25AnxA2 in nuclear subfractions of PC12 cells (A). Proteins (100 μg) from the nucleoplasm (lane 1) and NE (lane 2) fractions were separated by 10% SDS/PAGE and subjected to western blot analysis. The blot was probed with antibodies against pSer25AnxA2, total AnxA2, tubulin, SPC and fibrillarin, as indicated. myr‐ψ‐PKC inhibits Ser25 phosphorylation of AnxA2 and shifts the localisation of AnxA2 from the perinuclear to the cortical region of PC12 cells (B, C). Proteins (100 μg) from the cytoplasmic (lanes 1 and 2) and nuclear (lanes 3 and 4) fractions prepared from myr‐ψ‐PKC‐treated (+; lanes 2 and 4) and control (−; lanes 1 and 3) cells were separated by 10% SDS/PAGE and subjected to western blot analysis (B). The blots were probed with antibodies against pSer25AnxA2, total AnxA2, tubulin and GAPDH, as indicated. Detection of the resulting protein bands (A, B) was performed using the ChemiDoc™ XRS+ molecular imager after incubation with HRP‐conjugated secondary antibodies and ECL reagent. The arrows to the left indicate the protein molecular mass standards (nd, not determined). Immunofluorescence staining of control and myr‐ψ‐PKC‐treated cells, as indicated, was carried out using rabbit polyclonal antibodies against AnxA2 (green) (C). DNA staining with DAPI (blue fluorescence) was included to visualise the nucleus. Scale bar is 10 μm. The insets show higher magnification of the indicated areas in the images shown to the right in Panel (C). less

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Reactive oxygen species exert opposite effects on Tyr23 phosphorylation of the nuclear and cortical pools of annexin A2.

In J Cell Sci on 15 January 2016 by Grindheim, A. K., Hollås, H., et al.

Fig.5.A

Fig.5.A showing Western Blotting in a Rattus norvegicus (Rat) sample from the publication: Reactive oxygen species exert opposite effects on Tyr23 phosphorylation of the nuclear and cortical pools of annexin A2.

Incorporation of ubiquitylated pTyr23AnxA2 into extracellular vesicles (exosomes) and decrease in the cortical pool of pTyr23AnxA2 after prolonged treatment of cells with H2O2. (A) PC12 cells were grown in exosome-depleted medium in the presence of H2O2 for 0 min (lanes 1,5), 15 min (lanes 2,6), ... more

Incorporation of ubiquitylated pTyr23AnxA2 into extracellular vesicles (exosomes) and decrease in the cortical pool of pTyr23AnxA2 after prolonged treatment of cells with H2O2. (A) PC12 cells were grown in exosome-depleted medium in the presence of H2O2 for 0 min (lanes 1,5), 15 min (lanes 2,6), 1 h (lanes 3,7), or 2 h (lanes 4,8). ECM-bound proteins were released by EGTA (lanes 1–4), whereas extracellular vesicles were isolated from the culture medium by the ExoQuick-TC method (lanes 5–8). 100 µg of protein from the EGTA-released fractions (lanes 1–4) and the control extracellular vesicle fraction (lane 5), or an equal volume of extracellular vesicles from H2O2-treated cells (lanes 6–8) were separated by 10% SDS-PAGE, transferred to nitro-cellulose membranes and probed with antibodies against pTyr23AnxA2, total AnxA2, CD63, TSG-101 and T-cadherin, as indicated. (B) Following 1 h treatment of PC12 cells with 1 mM H2O2, proteins (600 µg) present in purified extracellular vesicles were immunoprecipitated (IP) by monoclonal AnxA2 antibodies (lane 1) after pre-clearance of the samples with normal mouse IgG (lane 2). The proteins were subjected to 10% SDS-PAGE and immunoblot analysis using monoclonal antibodies against pTyr23AnxA2 or ubiquitin by loading half of the immunoprecipitation sample on each gel. The bands representing ubiquitylated AnxA2 (square bracket; Ub-AnxA2) and IgG light chain (LC, arrowhead) are indicated to the right. (A,B) Following incubation with HRP-conjugated secondary antibodies and the ECL-reagent, the reactive protein bands were detected using the ChemiDoc™ XRS+ molecular imager. Note that the secondary antibody used in B only recognises the IgG light chains. Molecular mass standards are indicated to the left. (C) PC12 cells were untreated (C1), or treated for 15 min (C2), 30 min, (C3), 60 min (C4) or 120 min (C5) with 1 mM H2O2. The localisation of pTyr23AnxA2, detected using the monoclonal antibody (green), is shown in the merged images, which also display nuclear staining (DAPI, blue). Scale bars: 10 µm. less

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Reactive oxygen species exert opposite effects on Tyr23 phosphorylation of the nuclear and cortical pools of annexin A2.

In J Cell Sci on 15 January 2016 by Grindheim, A. K., Hollås, H., et al.

Fig.5.B

Fig.5.B showing Immunoprecipitation in a Rattus norvegicus (Rat) sample from the publication: Reactive oxygen species exert opposite effects on Tyr23 phosphorylation of the nuclear and cortical pools of annexin A2.

Incorporation of ubiquitylated pTyr23AnxA2 into extracellular vesicles (exosomes) and decrease in the cortical pool of pTyr23AnxA2 after prolonged treatment of cells with H2O2. (A) PC12 cells were grown in exosome-depleted medium in the presence of H2O2 for 0 min (lanes 1,5), 15 min (lanes 2,6), ... more

Incorporation of ubiquitylated pTyr23AnxA2 into extracellular vesicles (exosomes) and decrease in the cortical pool of pTyr23AnxA2 after prolonged treatment of cells with H2O2. (A) PC12 cells were grown in exosome-depleted medium in the presence of H2O2 for 0 min (lanes 1,5), 15 min (lanes 2,6), 1 h (lanes 3,7), or 2 h (lanes 4,8). ECM-bound proteins were released by EGTA (lanes 1–4), whereas extracellular vesicles were isolated from the culture medium by the ExoQuick-TC method (lanes 5–8). 100 µg of protein from the EGTA-released fractions (lanes 1–4) and the control extracellular vesicle fraction (lane 5), or an equal volume of extracellular vesicles from H2O2-treated cells (lanes 6–8) were separated by 10% SDS-PAGE, transferred to nitro-cellulose membranes and probed with antibodies against pTyr23AnxA2, total AnxA2, CD63, TSG-101 and T-cadherin, as indicated. (B) Following 1 h treatment of PC12 cells with 1 mM H2O2, proteins (600 µg) present in purified extracellular vesicles were immunoprecipitated (IP) by monoclonal AnxA2 antibodies (lane 1) after pre-clearance of the samples with normal mouse IgG (lane 2). The proteins were subjected to 10% SDS-PAGE and immunoblot analysis using monoclonal antibodies against pTyr23AnxA2 or ubiquitin by loading half of the immunoprecipitation sample on each gel. The bands representing ubiquitylated AnxA2 (square bracket; Ub-AnxA2) and IgG light chain (LC, arrowhead) are indicated to the right. (A,B) Following incubation with HRP-conjugated secondary antibodies and the ECL-reagent, the reactive protein bands were detected using the ChemiDoc™ XRS+ molecular imager. Note that the secondary antibody used in B only recognises the IgG light chains. Molecular mass standards are indicated to the left. (C) PC12 cells were untreated (C1), or treated for 15 min (C2), 30 min, (C3), 60 min (C4) or 120 min (C5) with 1 mM H2O2. The localisation of pTyr23AnxA2, detected using the monoclonal antibody (green), is shown in the merged images, which also display nuclear staining (DAPI, blue). Scale bars: 10 µm. less

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