Secondary anti rabbit igg

Cells were plated at a density of 5 × 10 5 per well in six-well plates. The following day, cells were treated with drug or equal volume of DMSO for 6, 24, and 48 h. Cell extracts were prepared by adding 2x Laemmli sample buffer supplemented with a phosphatase and protease inhibitor cocktail. Lysates were sonicated and the protein concentration was quantified using bicinchoninic acid (BCA) kit (Thermo, MA, USA). Equivalent protein was loaded and proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis then transferred to 0.45 µm nitrocellulose membranes. Equivalent loading was confirmed by staining membranes with Ponceau S dye. Membranes were blocked for 1 h in blocking buffer (5% milk, 1× TBS, 0.1% Tween 20) and placed in primary antibody (5% bovine serum albumin, 1× TBS, 0.1% Tween 20) overnight at 4°C. The following day, membranes were washed thrice in wash buffer (1× TBS, 0.1% Tween 20). Primary antibodies against Akt, p-Akt, p53, p-p53, EGFR, p-EGFR, p27, BIM, BAX, cyclin D1, c-Myc, GAPDH, secondary antirabbit IgG, and anti-mouse IgG were purchased from Cell Signaling Technologies (Danvers, MA, USA). Proteins were detected using horseradish peroxidase-linked secondary antibodies and visualized with the enhanced chemiluminescent detection system (GE Healthcare Biosciences, Pittsburgh, PA, USA). Immunoblot experiments were performed at least twice.