Anti vinculin 7f9 sc 73614

Cells were fixed with 4% paraformaldehyde for 10 min at 4 °C and washed twice for 10 min with PBS. The cells were permeabilized for 30 min using PBS, 3% BSA, and 0.1% Triton X-100, followed by anti-vinculin (7F9) sc-73614, anti-integrin-β1 sc-8978 (all from Santa Cruz Biotechnology10410 Finnell Street Dallas, Texas 75220 U.S.A.), anti-cytokeratin 8 (DC10) (from Thermo Fisher Scientific 75 Panorama Creek Dr, Rochester, NY 14625, USA), and anti-α-tubulin T5168 (from Sigma Aldrich 3050 Spruce St. Louis, MO, USA). Staining in PBS and 3% BSA was conducted at 4 °C overnight. The cells were washed with PBS and incubated for 1 h at room temperature with the appropriate secondary antibody FITC conjugated (Invitrogen Molecular Probes Eugene, OR, USA). Negative controls were processed in the same conditions as those used in primary antibody staining. For F-actin visualization, rhodamine phalloidin (Invitrogen Molecular Probes Eugene, 1: 40 dilution) was used. Cells were then washed in PBS and mounted in buffered glycerol (0.1 M, pH 9.5). Finally, analysis was conducted using a Leica confocal microscope TCS SP2 (Leica Microsystems Heidelberg GmbH, Mannheim, Germany) equipped with Ar/ArKr and He/Ne lasers. Laser line were at 543 and 488 nm for TRITC and FITC excitation, respectively. The images were scanned under a 40 × oil objective.

Control and stimulated cells were washed twice with ice-cold PBS and scraped in RIPA lysis buffer (Sigma Aldrich). A mix of protease inhibitors (Complete-Mini Protease Inhibitor Cocktail Tablets, Roche, Mannheim, Germany) and phosphatase inhibitors (PhosStop; Roche, Mannheim, Germany) was added just before use. Cellular extracts were then centrifuged at 8000× g for 10 min. The Bradford assay was used to determine protein contents. For western blot analysis, cellular extracts were separated on SDS-polyacrylamide gels and proteins were blotted onto nitrocellulose membranes (BIO-RAD, Bio-Rad Laboratories, Hercules, CA, USA). The following antibodies were analyzed: anti-vinculin (7F9): sc-73614; anti-Rock1 (H-85): sc-5560 and anti-beta-catenin sc-7963 all from Santa Cruz Biotechnology; anti-p53 (acetyl k382) ab-75754 from Abcam; anti-TPT1 (E-AB-31729) from Elabscience; anti-E-cadherin (610181) from BD Bioscience. Antigens were detected with an enhanced chemiluminescence kit (Western Bright ECL HRP Substrate, Advansta Inc., Menlo Park, CA, USA), according to the manufacturer’s instructions.