Immunoprecipitation of β3 Integrin

Adherent cells were washed twice in cold phosphate-buffered saline and lysed in RIPA buffer (Thermofisher, 89900) (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% Nonidet, P-40, 0.5% sodium deoxycholate and 0.1% sodium dodecyl) with freshly added protease inhibitor mixture (Roche, 5056489001). Cells debris were removed by centrifugation at 9000 g for 20 min at 4 °C. A micro BCA assay was used to quantify protein concentration. Cell lysates were precleared with dynabeads protein G magnetic beads (Thermofisher, 10003D). Immunoprecipitation was performed overnight at 4 °C with 40 µl of dynabeads protein G that were preincubated with 8 µL of a rabbit anti-β3 integrin subunit antibody (Sigma, AB2984, 1 mg/ml). After washing the beads with the lysis buffer, immunoprecipitated proteins were eluted with a SDS sample buffer under non-reducing conditions. They were analyzed by western blotting after separation on 10% SDS-polyacrylamide gel electrophoresis using the mouse monoclonal anti-CD47 antibody (B6H12, Santa Cruz, sc-12730, dilution 1:200) and a horseradish peroxidase-conjugated secondary antibody for chemiluminescence detection (Sigma, A0545, dilution 1:3000).