Rabbit polyclonal anti fak

KS1767 cells were seeded (1 × 10⁶) into 6 well plates and grown to 80% confluence. Cells were incubated with each peptide (100 µM) for 10, 30 and 60 min, followed by protein extraction in 50 mM Tris, 150 mM NaCl, 1% NP-40, phosphatase/protease inhibitors (Roche). Protein extracts were resolved by SDS-PAGE, transferred to nitrocellulose membranes and decorated with standard protocols. The following primary antibodies (all Cell Signaling) were used at a 1:1,000 dilution: rabbit polyclonal anti-Fak (#3285), rabbit monoclonal anti-phosphorylated Fak (Tyr³⁹⁷, #8556), mouse monoclonal anti-Akt (2H10), rabbit monoclonal anti-phosphorylated Akt (Ser⁴⁷³, D9E), rabbit monoclonal anti-phosphorylated p44/p42 (Thr²⁰²/Tyr²⁰⁴), and mouse monoclonal anti-Erk1/2 (p44/p42) (3A7). The rabbit anti-actin antibody (Sigma) was used at a 1:2,000 dilution.

Immunoblotting was performed exactly as previously described (6 (link), 42 (link)), using the following primary antibodies (1:1,000): rabbit polyclonal anti-phospho FAK Y925 (Cell Signaling, Beverly, MA, USA), rabbit polyclonal anti-FAK (Cell Signaling), rabbit monoclonal anti-human TCF4 (Cell Signaling), mouse anti-E-cadherin (1:2,000; BD Biosciences), mouse anti-human α-Tubulin (Sigma-Aldrich) and appropriate secondary antibodies (diluted 1:5,000): HRP-conjugated goat-anti-mouse or goat-anti-rabbit IgG (Merck-Millipore, Darmstadt, Germany). For immunoprecipitation, cell lysates of Caco2 cells were prepared 72 h after transfection with control or Sdc-1 siRNA as described previously (42 (link)). 0.5 mg protein was incubated with 1:50 dilution of primary antibody (rabbit monoclonal anti-human EGR1, Cell Signaling) at 4°C on a rocker platform overnight. Afterward, the mixture was incubated analogously with 20 μl resuspended protein A/G-PLUS-Agarose. Immunoprecipitates were pelleted by centrifugation (1,000 g, 5 min, 4°C), washed four times with RIPA buffer and boiled in 40 μl SDS sample buffer (5 min). SDS-PAGE, Western blotting, stripping and reprobing were performed as described previously (6 (link)) using 30–60 μg of protein/lane on 7.5– 12% gels.