Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Primary antibodies raised against human S100A11 (1:1000 dilution) (ProteinTech Group), Annexin A1 (1:1000 dilution) (Abcam), Annexin A2 (1:1000 dilution) (BD Transduction Laboratories), heat shock cognate 70 kDa protein (1:6000 dilution) (Hsc70; N69, kindly provided by Boris Margulis, Russian Academy of Sciences, St. Petersburg, Russia), α-tubulin (1:5000) (Abcam) and ErbB2 (1:1000) (Fischer Thermo Scientific, MA) was used. This was followed by appropriate peroxidase-conjugated secondary antibodies (DAKO). Immunocytochemistry: cells on coverslips were fixed in paraformaldehyde and stained with indicated primary antibodies (1:300 dilution) including S100A11 and β-actin (Sigma). Samples were incubated with the appropriate Alexa Fluor-488– and Alexa- Fluor-546/594-coupled secondary antibodies (1:1000 dilution) (Molecular Probes) and images taken by confocal microscopy. Immunoprecipitation (IP) was performed on lysates from HeLa or MCF7-ErbB2 cells overexpressing ANXA2-RFP and S100A11-GFP (wild type or mutants) or only S100A11-turbo-GFP. Immuno complexes were captured with RFP-Trap agarose beads (Chromotek) or turbo-GFP antibody (OriGene) coupled to sepharose-G beads and washed 4 times before immunoblot analysis (Extended blots showing Co-IP are presented in Supplementary Figure 4 ).
Annexin a2
Manufactured by BD
Sourced in Sao Tome and Principe, United Kingdom
Annexin A2 is a protein that plays a role in the regulation of cellular processes. It is a member of the annexin family of calcium-dependent phospholipid-binding proteins. Annexin A2 is involved in several cellular functions, including membrane organization, vesicle trafficking, and signal transduction.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (5 mentions)
Peroxidase conjugated secondary antibody, by Agilent Technologies (2 mentions)
α tubulin, by Abcam (2 mentions)
Annexin a1, by Abcam (2 mentions)
Raised against human s100a11, by Proteintech (2 mentions)
Alexa fluor 488 and alexa fluor 546 594 coupled secondary antibodies, by Thermo Fisher Scientific (2 mentions)
Rfp trap agarose beads, by Proteintech (2 mentions)
Turbo gfp antibody, by OriGene (2 mentions)
Image studio 5, by LI COR (2 mentions)
Phospo tyr24 annexin a2, by Santa Cruz Biotechnology (2 mentions)
Odyssey sc, by LI COR (2 mentions)
Annexin a2, by Abcam (2 mentions)
E cadherin, by Abcam (1 mentions)
Fibronectin, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Merck Group (1 mentions)
Vimentin, by Merck Group (1 mentions)
Annexin 2, by BD (1 mentions)
N cadherin, by BD (1 mentions)
E cadherin, by BD (1 mentions)
5 protocols using annexin a2
1
Immunological Analysis of Protein Interactions
2
Immunological Analysis of Protein Interactions
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Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Primary antibodies raised against human S100A11 (1:1000 dilution) (ProteinTech Group), Annexin A1 (1:1000 dilution) (Abcam), Annexin A2 (1:1000 dilution) (BD Transduction Laboratories), heat shock cognate 70 kDa protein (1:6000 dilution) (Hsc70; N69, kindly provided by Boris Margulis, Russian Academy of Sciences, St. Petersburg, Russia), α-tubulin (1:5000) (Abcam) and ErbB2 (1:1000) (Fischer Thermo Scientific, MA) was used. This was followed by appropriate peroxidase-conjugated secondary antibodies (DAKO). Immunocytochemistry: cells on coverslips were fixed in paraformaldehyde and stained with indicated primary antibodies (1:300 dilution) including S100A11 and β-actin (Sigma). Samples were incubated with the appropriate Alexa Fluor-488– and Alexa- Fluor-546/594-coupled secondary antibodies (1:1000 dilution) (Molecular Probes) and images taken by confocal microscopy. Immunoprecipitation (IP) was performed on lysates from HeLa or MCF7-ErbB2 cells overexpressing ANXA2-RFP and S100A11-GFP (wild type or mutants) or only S100A11-turbo-GFP. Immuno complexes were captured with RFP-Trap agarose beads (Chromotek) or turbo-GFP antibody (OriGene) coupled to sepharose-G beads and washed 4 times before immunoblot analysis (Extended blots showing Co-IP are presented in Supplementary Figure 4 ).
3
Annexin A2 Immunoblotting Analysis
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The cells were lysed in 1% NP-40 lysis buffer then quantified and denatured with SDS loading buffer and boiled for 5 min. Lysates were separated on 12% SDS acrylamide gels and subsequently transferred to nitrocellulose membrane. Membranes were blocked using 5% BSA for 1 h at room temperature then probed with corresponding primary antibodies at 4 °C overnight. Dilutions of antibodies were as follows: Annexin A2 1:1000 (Abcam, Cambridge UK), Annexin A2 1:1000 (BD Bioscience), B-Actin 1:1000 (Sigma), phospo-Tyr24-Annexin A2 1:250 (Santa Cruz Biotechnology, Heidelberg, Germany) E-Cadherin 1:1000 (Abcam). IRdye700- or IRdye800-conjugated secondary antibodies, were then coupled to the primary antibody for 1 h at room temperature. Protein bands were detected using the Odyssey Sc (LI-COR, Cambridge, UK).and quantified using Image Studio 5.2 (LI-COR)).
4
Annexin A2 Protein Quantification
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The cells were lysed in 1% NP-40 lysis buffer then quantified and denatured with SDS loading buffer and boiled for 5 min. Lysates were separated on 12% SDS acrylamide gels and subsequently transferred to nitrocellulose membrane. Membranes were blocked using 5% BSA for 1 h at room temperature then probed with corresponding primary antibodies at 4°C overnight. Dilutions of antibodies were as follows: Annexin A2 1:1000 (Abcam, Cambridge UK), Annexin A2 1:1000 (BD Bioscience), B-Actin 1:1000 (Sigma), phospo-Tyr24-Annexin A2 1:250 (Santa Cruz Biotechnology, Heidelberg, GE) GAPDH 1:1000 (Sigma) Na/K-ATPase 1:500 (Cell Signaling, Beverly, MA), Fibronectin 1:100, (Santa Cruz). IRdye700- or IRdye800-conjugated secondary antibodies, were then coupled to the primary antibody for 1 h at room temperature. Protein bands were detected using the Odyssey Sc (LI-COR, Cambridge, UK) and quantified using Image Studio 5.2 (LI-COR).
5
Comprehensive Protein Expression Analysis
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β-actin (Sigma Aldrich mouse monoclonal anti-β-actin, A2228, 1:2000), 42 kDa. N-cadherin (BD Biosciences mouse monoclonal anti-N-cadherin, 610921, 1:2000) 120 kDa. E-cadherin (BD Biosciences mouse monoclonal anti-E-cadherin, 610181, 1:2000) 120 kDa. Vimentin (Sigma-Aldrich goat polyclonal anti-Vimentin, V4630, 1:1000) 58 kDa. S100A10(BD Biosciences mouse monoclonal anti-S100A10, 610070, 1:2000) 11 kDa. Annexin A2 (BD Biosciences mouse monoclonal anti-Annexin II, 610069, 1:2000) 36 kDa. GAPDH (Biochain mouse monoclonal anti-GAPDH, Y3322, 1:2000) 36 kDa. p-S6K (Cell signaling rabbit monoclonal anti-pS6K, 9205 S, 1:1000) 70 kDa. FOXC2 (Bethyl laboratories rabbit polyclonal anti-FOXC2, A302-383A, 1:1000) 65–70 kDa. PAI-1 (Cell signaling rabbit monoclonal anti-PAI-1 D9C4, 11907, 1:2000) 48 kDa. uPAR (Santa Cruz rabbit polyclonal anti-uPA H149, sc-10815, 1:300) 55 kDa. p-ERK (Erk1/2) (Cell signaling rabbit polyclonal anti-Erk1/2 (Thr202/Tyr204), 9101, 1:1000) 42, 44 kDa.
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