Total akt

Western blotting was conducted as described [23 (link)]. Briefly, whole cell extract was prepared with lysis buffer (10 mM Tris, pH 7.5, 0.1 mM EDTA, 0.1 mM EGTA, 0.5% SDS, 0.1 mM ß-mercaptoethanol, containing 2 μg/ml of each of the protease inhibitors leupeptin, aprotinin, and pepstatin). We resolved lysates on 8~15% SDS-PAGE and immunoblotted the nitrocellulose membrane with the antibodies. The blot was then detected by chemiluminescence (ECL, Amersham Pharmacia Biotech. Arlington Heights, IL). Antibodies against RhoB, total-Akt, phospha-Akt were from Santa Cruz Biotechnology, ß-actin was from Sigma-Aldrich Chemicals, and antibodies against total-p38, JNK, ERK or phospho-p38, JNK and ERK were purchased from Cell signal Technology.

Non-small cell lung cancer (HCC827, H1975) cell lines were treated with the tested drug candidates for the designated time (4 h). The cells were then harvested for Western blot analysis in lysis buffer (0.05-M HEPES pH 7.4, 0.15-M NaCl, 2-mM EDTA, 10% v/v glycerol, 1% v/v Triton X-100) supplemented with protease and phosphatase inhibitor cocktail (Thermo Scientific). Whole cell lysates were separated by SDS-PAGE and subjected to immunoblot analysis with the respective antibodies [total EGFR, phosphor-EGFR (Y845), phosphor-ERK1/2 (Thr177/Thr160), ERK1/2, phosphor-Akt, total Akt, and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA)]. Primary antibody incubation was carried out at 4°C overnight in 5% bovine serum albumin/phosphate-buffered saline-Tween 20. Afterward, the membranes were incubated with HRP-conjugated donkey anti-mouse/anti-rabbit secondary antibody at room temperature for 1 h, and developed using the WesternBright Quantum chemiluminescence detection system (Advansta Corporation, Menlo Park, CA, USA). Anti-GAPDH antibody was used as the loading control (Santa Cruz Biotech, Santa Cruz, CA, USA). Digital chemiluminescence images were captured and quantified by using the FluorChem Q Imaging System (Alpha Innotech Corporation, Santa Clara, CA, USA).