EGFR-Akt Signaling Modulation

CM cells were plated in 6-well plates. Twenty-four hours after plating, cells were added with DMSO or 20 µg/ml cetuximab, 2.5 µM gefitinib, with or without 20 ng EGF, and were analyzed following 24h incubation. Whole cell lysate preparation and western blotting were performed as previously described (104 (link)). Ten µg cell lysate were separated on Criterion TGX 4-15% gradient gels (BioRad) and blotted onto nitrocellulose membranes. Immunoblotting was performed using the following antibodies, under the manufacturer’s instructions: anti-phospho-Akt (Ser473) (#9271), -total-AKT (#9271), -phospho-EGFR (Tyr1068) (#3777), and -total-EGFR (#2232) antibodies from Cell Signaling Technology; anti-β-tubulin (H-235) (sc-9104), used as a loading control, from Santa Cruz Biotechnology. Revelation was performed using the Clarity Western ECL Substrate (BioRad) or the SuperSignal Femto reagent (Pierce) through Chemidoc XRS+ instrument (Biorad). Image analysis was performed with the Image Lab v6.1 (Biorad) software. The Student’s t-test for two tailed distributions was used to compare data. Differences were considered statistically significant when p≤ 0.05.