Anti fak antibody

Immunoprecipitation and kinase reactions were performed as previously described [32 (link)]. Briefly, equal amounts of total cell lysate (600 μg) were immunoprecipitated for FAK with anti-FAK antibody (BD Bioscience, Mississauga, ON) and 20 μl of Protein A/G sepharose beads (GE Healthcare, Uppala, Sweden) while rotating for 2 hours at 4°C. The beads were then washed 3 times with 200 mM NETN (20 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0, 200 mM NaCl, 0.5% Igepal CA-630) followed by one wash in kinase buffer (0.25 mM NaVO₃, 20 mM Tris-HCl, pH 7.4, 1 mM NaF, 10 mM β-glycerophosphate, 1 mM dithiothreitol, 15 mM MgCl₂). DMSO (vehicle control) or FAK inhibitor PF-228 at 1 or 5 μM concentrations were then added to the reaction in 20 μl of kinase buffer and incubated for 20 minutes at 30°C. The kinase assay was then initiated with 1 μl of [³²P] γATP (5 μCi/μl) and incubated for 30 minutes at 30°C with mixing every 10 minutes. The reaction was terminated following the addition of 4X SDS sample buffer. The samples were resolved on a 7.5% polyacrylamide gel and transferred to PVDF membrane. The membrane was subjected to autoradiography for development of FAK phosphorylation events and probed for total FAK levels by western blotting as described below.

Proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were blocked with PBST with 2.5% skim milk, incubated with anti-FAK(p397) antibody (Abcam), anti-FAK antibody (BD Biosciences), anti-N-cadherin antibody (BD Biosciences), or anti-β-actin antibody (Sigma-Aldrich, St. Louis, MO, USA), and then with horseradish peroxidase-coupled secondary antibody (Agilent, Santa Clara, CA, USA), and visualized with the ECL Western Blotting Detection Reagents (GE Healthcare, Chalfont St. Giles, UK).