Fatty Acid Oxidation Assay with SIRT3 Modulation

Antibodies used were specific for ATPase subunit α and β (Invitrogen Molecular Probes), monoclonal and polyclonal acetyllysine (Cell Signaling Technology), SIRT3 (as described3 (link)), ETF and LCAD (generously provided by Jerry Vockley, University of Pittsburgh). Oxidation of [1-¹⁴C] palmitic acid by tissue homogenate was adapted from a previously established method26 (link). Briefly, tissue was homogenized in sucrose/Tris/EDTA buffer, and was incubated for 30–60 min in the reaction mixture (pH 8.0), containing [1-¹⁴C] palmitic acid, and measured for acid-soluble metabolites (ASM) and trapped CO₂. Enzymatic activity for LCAD was measured using the anaerobic electron transfer flavoprotein (ETF) fluorescence reduction assay27 (link) using 2, 6-dimethyheptanoyl-CoA as a substrate in recombinant LCAD expressed and purified from HEK293T cells with wild-type or catalytically inactive SIRT3, or in E. coli in the absence (Control) or presence of nicotinamide (NAM, 50 mM)28 (link).