Vinculin antibody

We separated proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detected various proteins by Western blotting, as described previously [42] (link). The following primary antibodies were used for detection: anti-COX-2 monoclonal antibody (Cayman Chemical, Ann Arbor, MI), anti-GRHL2 (Sigma-Aldrich, St. Louis, MO), anti-FTO (EMD Millipore, Billerica, MA), anti-ZEB1, anti-histone H3, anti-histone H3 tri-methyl Lys-4, anti-histone H3 acetyl Lys-14, and anti-vimentin antibodies (Cell Signaling, Danvers, MA). We used the ECL prime blocking agent (GE Healthcare Life Sciences, Piscataway, NJ) for blocking and Lumigen TMA-6 reagents for detection (Lumigen, Inc., Southfield, MI). The filters were stripped by incubating the membrane in 0.5% Triton X-100 and were re-probed with a monoclonal β-actin antibody (Sigma-Aldrich, St. Louis, MO) or with vinculin antibody (Abcam, Cambridge, MA), which served as gel-loading controls. To detect β-actin or vinculin, we used 2% non-fat dry milk (Bio-Rad, Hercules, CA) for blocking and ECL prime reagent for detection (GE Healthcare Life Sciences, Piscataway, NJ). We performed each western blot at least twice; the representative blots are shown. We quantified the protein bands on x-ray films by using the ImageJ image processing program (National Institutes of Health).

Cells were seeded on round-coverslips coated with 1:100 fibronectin in PBS (overnight, 4 °C) at the concentration 2 × 10⁴ cells/mL (1 mL per six-well plate) 32 h after transfection. The cells were fixed 24 h after seeding with 3.7% Formalin and permeabilized using 0.1% Triton-X-100 and blocked using 1:10 BSA Aurion in PBS. The antibodies were diluted in 1% BSA/PBS solution and stained for 24 h. The vinculin antibody (polyclonal, rabbit, Abcam, Cambridge, UK) was diluted at 1:300. Each coverslip was then washed three times with PBS. The secondary antibody goat-anti-rabbit IgG Alexa Fluor^® 488 (1:500, Invitrogen, Eugene, OR, USA) was diluted 1:500 in 1% BSA/PBS, and Alexa Fluor 568 Phalloidin was used to stain F-actin at 1:1000. The nuclei were stained using DAPI (1:2000) in 1% BSA/PBS for 5 min.