Western Blotting Analysis of Signaling Proteins

Cell lysate was prepared for western blotting as previously described [37 (link)]. Protein concentrations were determined using the Pierce BCA Assay (Thermo Scientific, Waltham, MA). Membranes were immunoblotted overnight with the following primary Abs: rabbit anti-human PTEN Ab, rabbit anti-human p85 Ab, rabbit anti-human phospho-p85 Ab, rabbit anti-human p110-alfa Ab, rabbit anti-human p110-beta Ab, rabbit anti-human p110 gamma Ab (1:1000, Cell Signaling, Danvers, MA), rabbit anti-human c-Met Ab (1:250, Life technologies), rabbit anti-human phosphor-c-Met Ab (p-c-Met), rabbit anti-human total-Akt Ab (1:500, Cell Signaling), rabbit anti-human phospho-Akt (p-Akt) Ab (Ser473, 1:100, Cell Signaling), mouse anti-human CDKN1A Ab (1:200, BD Pharmingen) and mouse anti-human β-actin Ab (loading control; 1:5000, Cell Signaling). After immunoblotting, the membranes were washed with 1X TBS containing 0.1% Tween-20, followed by a 1-hr incubation with horseradish peroxidase-conjugated rabbit anti-mouse Ab (1:5000, Santa Cruz Biotechnology) and goat anti-rabbit Ab (1:5000, Santa Cruz Biotechnology). Immunoreactive bands were visualized with the Super Signal West Dura Extended Substrate kit (Thermo Scientific) and the densities of the protein bands were quantified by the Alpha-Ease FCTM software (version 3.1.2, Alpha Innotech, San Leandro, CA).