Western blotting

Western blot analysis was performed, as described previously [44]. Briefly, whole-cell lysates were prepared in a modified RIPA buffer containing proteinase inhibitors and phosphatase inhibitors as described elsewhere [45]. Equivalent amounts of protein (20–80 μg) were loaded in 10% or 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels and transferred by blotting to polyvinylidene fluoride (PVDF) membranes. The blot was incubated with primary antibodies against pFAK (Tyr 925; Cell Signaling Technology), pFAK (Tyr 576/577: Cell signaling technology), total FAK, pAKT, AKT, pERK1/2, ERK1/2 (Cell Signaling Technology), and actin (Santa Cruz Biotechnology, Inc.). After washing, the blot was incubated with HRP-conjugated secondary antibodies. The protein–antibody complexes were detected using enhanced chemiluminescence (Amersham, Arlington Heights, IL, USA), according to the manufacturer's recommended protocol.

Western blot bands were quantified using Image J software (NIH, Bethesda, MD, USA). The adjusted relative densities were calculated relative to the expression of control sample's actin