Signaling Pathway Protein Analysis

Whole cell lysates were prepared in NP-40 lysis buffer supplemented with a protease inhibitor cocktail (Sigma, St. Louis, MO) and Halt phosphatase inhibitor cocktail (Thermo Scientific, Waltham, MA). Supernatant was collected after centrifugation at 13,000 rpm for 10 min at 4°C and separated by SDS-PAGE. Anti-GIPC, anti-PLCγ, and the horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology. Antibodies against ABCG2, mTOR, phospho-mTOR, p70S6K, phospho-p70S6K, Atg7, Beclin1, AMPK-α, and phospho-AMPK-α were purchased from Cell Signaling Technologies. Anti-CHMP4b and anti-TSG101 was purchased from Abcam; anti-β-actin was purchased from Sigma; and the Alix antibody was purchased from Thermo Scientific. Western blots were developed using the SuperSignal West Pico substrate (Thermo Scientific) and immunoprecipitations were performed as previously described [19] (link).