Ecl western blotting solution

For in vitro studies, cSCC cells were treated as indicated of each individual experiment. Cells were rinsed with PBS and lysed in buffer containing Triton X-100 (1%), TrisHCL (0.05M), NaCl (0.1M) and protease/phosphatase inhibitors and EDTA (5mM). Lysates were centrifuged to extract protein at 15,000 rpm. Protein was quantified using Bradford analysis followed by denaturing with sample buffer containing a reducing agent. Proteins were separated on a 4–12% gradient gel, transferred on PVDF membrane, blocked in Non-fat milk (5%) and incubated in primary antibodies overnight at 4 degrees. Blots were rinsed, incubated with the 2nd antibody and developed using ECL western blotting solution (BioRad). Protein levels of AKT, pAKT, S6, pS6, p70S6K, pp70S6K were all determined by Western blotting using antibodies from Cell Signaling Technology (Danvers, MA). Levels of FGFR2 was detected using anti-FGFR2 (sc-53219, Santa Cruz Biotechnology, Santa Cruz, CA) antibody. Primary antibody against β-actin was purchased from Abcam (Cambridge, MA).