Quantitative Western Blot Analysis of Protein Expression

Western blotting to analyze protein expression was performed as previously described^{62 –64 (link)}. Briefly, cell pellets and tissues were lysed using RIPA lysis buffer (Millipore) and the protein concentration was determined by a Bradford assay (Bio-Rad) using a Nanodrop2000 (Thermo scientific). 15–20 μg of protein was used for immunoblot analysis using antibodies against phospho-AMPKα (Thr172, 1:1000, 2535, Cell Signaling), AMPKα (1:1000, 5832, Cell Signaling) AMPK α1 (1:1000, 2795, Cell Signaling), AMPKα2 (1:1000, 2757, Cell Signaling), phospho-ACC (S79, 3661, 1:1000, Cell Signaling), ACC (1:1000, 3662, Cell Signaling), phospho-S6 (Ser240/244, 1:5000, 3661, Cell Signaling), S6 (1:1000, 2217, Cell Signaling), phospho-S6K (Thr389, 1:1000, 9205, Cell Signaling), S6K (1:1000, sc-230, Santa Cruz), phospho-ULK1 (S757, 6888, 1:1000, Cell Signaling), ULK1 (1:1000, 8054, Cell Signaling), LKB1 (1:1000, sc-32245, Santa Cruz), SLC7A11 (1:3000, 12691, Cell Signaling), GPX4 (1:1000, MAB5457, R&D systems), ACSL4 (1:1000, sc-271800, Santa Cruz), Cleaved Caspase-3 (Asp175, 1:500, 9661, Cell Signaling), Cleaved-PARP (Asp214, 1:1000, 9544, Cell Signaling), Vinculin (1:50000, V4505, Sigma).