Anti vinculin sc 7649

Cells were lysed in buffer containing 1% Nonidet P-40, 1 mmol/liter EDTA, 50 mmol/liter Tris-HCl (pH 7.5), and 150 mmol/liter NaCl supplemented with Complete protease inhibitors (Roche). Total proteins were resolved in a 10-12% polyacrylamide gel under denaturing conditions and transferred to nitrocellulose filters for Western blot analyses. Membranes were blocked with 5% BSA in TBS and incubated with the primary antibodies. Membranes were then incubated with the horseradish peroxidase–conjugated secondary antibody (1:3.000) and the reaction was detected with a Western blotting detection system (enhanced chemiluminescence; GE Healthcare). The primary antibodies used were: anti-PATZ1 [22 (link)]; anti-NESTIN (sc-23927, Santa Cruz Biotechnology, Santa Cruz, CA); anti-PDGFRβ (3169, Cell Signaling Technology, Boston, MA); anti-NANOG (sc-134218, Santa Cruz Biotechnology); anti-SOX2 (sc-20088, Santa Cruz Biotechnology); anti-CXCR4 (C8727, Sigma-Aldrich). To ascertain that equal amounts of protein were loaded, the membranes were incubated with anti-ACTIN (sc-1616-R, Santa Cruz Biotechnology) or anti-VINCULIN (sc-7649, Santa Cruz Biotechnology) antibodies.

Protein extraction and Western blotting were performed as previously described [28 (link),29 (link)]. The primary antibodies used were anti-Egr1 (ab191441) from Abcam (Cambridge, UK); anti-Gapdh (sc-32233) and anti-Vinculin (sc-7649) from Santa Cruz Biotechnology (Dallas, TX, USA); anti-α-Tubulin (t6074) from Sigma (St. Louis, MO, USA). Blots were visualized by using the Western blotting detection reagents (Thermo Fisher Scientific Inc., Waltham, MA, USA).