Western Blot and HDAC3 Assay Protocol

For western blot, tissues or cells were lysed in RIPA buffer, which contained PMSF and proteinase inhibitor. The concentration of total protein was quantified using Bradford protein assay. Lysates were resolved on SDS-PAGE gel electrophoresis and then transferred to PVDF membranes. The membranes were further treated with skimmed milk and blotted with antibodies of HDAC3 (Abcam 7030), Ampd3 (Abcam 194361), Hsp90 (CST 4874), Tubulin (CST 2148), and OXPHOS proteins (MitoSciences MS604). For HDAC assay, the HDAC3 protein was immunoprecipitated from the muscle protein lysates with 2 mg total protein, followed by a fluorescence-based HDAC assay reaction (Active Motif) performed with the pelleted beads (Sun et al., 2013 (link)).