Immunoblot and Immunoprecipitation Techniques

Immunoblots and immunoprecipitation were performed as previously describe (Han et al., 2020 (link); Wang et al., 2018 (link)). For immunoprecipitation analysis, 500 μg lysates were mixed with antibodies at 4°C 1 h followed by the addition of 40 μl of Protein A/G PLUS‐Agarose (Santa Cruz) at 4°C overnight. Immune complexes were washed three times with lysis buffer and then boiling. Equivalent amounts of protein samples (20 μg per lane) were subjected to SDS/PAGE. Total rodent OXPHOS cocktail antibody (ab110413) and rabbit polyclonal antibodies against SIRT1 (ab12193) from Abcam (MitoSciences‐Abcam, Eugene, OR). SIRT3 (#5490), AMP‐activated protein kinase α (AMPK α) (#2532), phosphor‐AMPKα (Thr¹⁷²) (#2535), and GAPDH (#2118), from Cell signaling and used according to protocols provided by the manufacturer.