Anti vinculin

Immunoblot analysis was performed as previously described (80 (link)). Briefly, cells were lysed in radioimmunoprecipitation assay buffer supplemented with a protease inhibitor cocktail (Roche). Whole-cell extracts were resolved by SDS–polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and probed with indicated primary antibodies. Bound antibodies were detected with horseradish peroxidase (HRP)–conjugated secondary antibodies and chemiluminescent HRP substrate. The following primary antibodies were used for Western blotting (all from CST, Beverly, MA, USA, unless otherwise indicated): anti-SOX9 (#82630; 1:1000), anti-vinculin (#13901; 1:1000), and anti-RFP (Rockland, 600-401-379; 1:500).

Cells were scraped from the wells and homogenized in RIPA buffer (Cell Signaling Technology) supplemented with 1× protease inhibitor (GenDepot) and 1× phosphatase inhibitor (GenDepot). The samples were processed, run on 4–20% Criterion TGX Stain-Free Precast Protein Gels (Bio-Rad Laboratories) and transferred as mentioned above. After blocking for 1 hr at room temperature with 5% Blotto Non Fat Dry-Milk (Chem Cruz) in PBST, membranes were probed overnight at 4°C with anti-FLAG (Sigma-Aldrich, M2, 1:2000), anti-Myc (Cell Signaling Technology, 71D10, 1:1000) or anti-vinculin (Sigma-Aldrich, nVIN-1, 1:5000) in 1:1 Odyssey Blocking Buffer PBS (Licor) in PBST. The next day, the blots were washed three times with PBST and incubated for 1 h at RT in secondary antibody. The secondary antibody used to detect anti-Myc was Rabbit IgG (H&L) Antibody DyLight 680 Conjugated (Rockland Immunochemicals; 1:10 000) in 5% non-fat milk in PBST. The secondary antibody used to detect anti-FLAG and anti-vinculin was Mouse IgG (H&L) Antibody DyLight 800 Conjugated (Rockland Immunochemicals; 1:10000) in 5% non-fat milk in PBST. The membranes were washed three times with PBST and then imaged using the Odyssey CLx Imaging System (Licor).