In vitro acetylation and deacetylation assay

The recombinant GST-GLDC and Flag-ACAT1 were purified and mixed in the buffer containing 40 mM Tris-HCl pH 8.0, 75 mM potassium chloride (KCl), and 10 μM acetyl CoA in a final volume of 30 μl. The reaction was incubated at 30 °C for 45 min and terminated by the addition of SDS-PAGE sample buffer and acetylated proteins were analyzed by immunoblots. For deacetylation assay, purified recombinant GST-GLDC and SIRT3 were incubated in 20 μl of deacetylation buffer (50 mM Tris–HCl pH 9.0, 4 mM MgCl₂, 50 mM NaCl, 0.5 mM dithiothreitol (DTT), 0.5 μM TSA) with or without 10 μM Ac-CoA, 1 mM NAD⁺, and NAM at 37 °C for 3 h with gentle agitation. For NAM treatment as a control, reactions were pretreated with 10 mM NAM for 10 min. The reaction was terminated by SDS/PAGE sample buffer, and the protein samples were subjected to immunoblotting analysis with the indicated antibodies.