Modified ripa lysis buffer

Whole cell extracts of H358-P and H358-S cells were prepared using modified RIPA lysis buffer (Merck Millipore, Billerica, MA,) containing protease inhibitors (Roche, Indianapolis, IN,) and phosphatase inhibitors (Thermo Scientific, Bremen, Germany). Western blot analysis was performed as previously described using 30 μg protein lysates [65 (link), 71 (link)]. Nitrocellulose membranes were hybridized with primary antibodies and developed using Luminol reagent (Santa Cruz Biotechnology, Dallas, TX,) as per the manufacturer's instructions. Anti-PAK6 antibody was obtained from Novus (Novus Biologicals, Littleton CO), Phospho-PAK6 (Ser 560), Anti-AKT and phospho-AKT (Ser 473) antibodies were obtained from Cell Signaling Technology (Cell Signaling Technology, Beverly, MA). Beta-actin antibody was obtained from Sigma (St. Louis, MO).

The H358-P and H358-S cells were lysed using modified RIPA lysis buffer (Merck Millipore, Billerica, MA,) containing protease inhibitors (Roche, Indianapolis, IN,) and phosphatase inhibitors (Thermo Scientific, Bremen, Germany). Western blot analysis was performed as described previously using 30 μg protein lysates [79 (link), 80 (link)]. Briefly, lysate were resolved and transferred on nitrocellulose membranes, which was further hybridized with primary antibodies and developed using Luminol reagent (Santa Cruz Biotechnology, Dallas, TX,). Anti-EGFR, phospho-EGFR (Y1068 and Y1197), anti-FAK, phospho-FAK (Y576/577), anti-SRC, and phospho-SRC (Y416) antibodies were obtained from Cell Signaling Technology (Cell Signaling Technology, Beverly, MA). Anti-FRK antibody was obtained from Santa Cruz Biotechnology. Beta-actin antibody was obtained from Sigma (St. Louis, MO). The FAK inhibitor PF-562271 was purchased from Santa Cruz Biotechnology (Dallas, TX). Erlotinib and Dasatinib were purchased from Selleckchem (Houston, TX). DMSO was used as vehicle in all experiments.

Whole cell extracts of GBC cells, were prepared using modified RIPA lysis Buffer (Merck Millipore, Billerica, MA) containing protease inhibitors (Roche, Indianapolis, IN) and phosphatase inhibitors (Thermo Scientific, Bremen, Germany). Rabbit polyclonal anti-MIF was obtained from Santa Cruz (sc-20121, Santa Cruz Biotechnology, Dallas, TX). β-Actin was used as a loading control. Western blot analysis was performed as previously described [32 (link)] using 30 μg protein lysates.