Assays were performed as described previously (Han et al., 2015 (link); Chen et al., 2017 (link)). In brief, cells were lysed in cold cell lysis buffer (HEPES pH 7.4 50 mmol/L, NaCl 150 mmol/L, Triton X-100 1% and glycerol 10%) supplemented with phosphatase and protease inhibitors (B14011 and B15001, Bimake), sonicated and centrifuged for 15 min at 15,000 rpm at 4 °C. Total protein (20 μg) from each lysate was separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and probed with the indicated antibodies. For immunoprecipitation, control IgG or antibody was incubated overnight at 4 °C with supernatant. Antibodies were purchased as follows: mouse monoclonal anti-HA antibody (MMS-101P), Covance; mouse monoclonal anti-FLAG antibody (F1804) and anti-Tubulin (T5201), Sigma-Aldrich; rabbit polyclonal anti-HA (561) and anti-FLAG (PM020), MBL International; anti-MFN2 (ab56889) and anti-COX4 (ab14744), Abcam; anti-PKM2 (4053), anti-S6K (2708s) and anti-pS6K (9234), Cell Signaling Technology; anti-Myc (SC-40), Santa Cruz. The phospho-S200 MFN2 antibody was generated and purified by AbMax Biotechnology.
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