Annexin a2

The presence of EV associated markers was confirmed as outlined in a previous publication.^{19 (link)}
Following electrophoretic separation using precast 4%–15% Mini-PROTEAN TGX gels (Biorad), the gels were blotted onto polyvinylidene difluoride (PVDF) membranes (Invitrogen) and blocked with 5% non-fat milk powder in Tris-buffered saline. After 1 h blocking at room temperature, the membranes were incubated with primary antibodies against Annexin A2 (37 kDa, Abcam, UK) and Alix (97 kDa, Santa Cruz, USA) in their respective blocking buffers overnight at 4 °C at a dilution of 1:1000 in Tris-buffered saline containing 0.5% Triton X 100. After washing, blots were incubated with 1:3000 horseradish peroxidase-conjugated secondary antibodies (Cell Signaling, UK) for 1 h at room temperature. Chemiluminescence detection of bands was performed with ECL Plus reagent (Biorad) and imaged using Image Lab Software (Life Science Research, BioRad, UK).

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)).

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).