Fig 1: HDACs 1, 2, and 3 catalyze lysine lactylation in vitro(A) Western blots of an in vitro lysine lactylation assay with recombinant HDAC1 (rHDAC1), rHDAC2, or rHDAC2/NcoR2 and histone H3 (rH3) in the presence of 1 mM L-Lactate with or without TSA (5 μM). The reactions were performed at 37°C for 30 mins. Protein loading was visualized by ponceau S staining, and Kbhb was detected by western blot. Experiments were repeated independently twice with similar results. (B-C) LC-MS/MS analysis of H3 after in vitro lysine lactylation assay. Detected Kla-modified sites on H3 (B). MS/MS-spectra for the indicated peptide with detected y and b ions (C). (D) Schematic of experimental workflow. HDAC2 KO HEK293T cells expressing 3xFLAG-mHDAC2 were used for immunoprecipitation with α-FLAG antibody. The immunoprecipitants were used for in vitro lysine lactylation assays and in vitro deacetylation assays. (E) Representative western blots of in vitro lysine lactylation with IPed 3xF-mHDAC2 WT or indicated mutants. This experiment was independently repeated three times with similar results. (F) Lysine lactylation and deacetylation activity for each mutant. Data are represented as mean +/− SEM activity relative to WT 3xF-mHDAC2, from n=3 independent experiments. (G) Schematic of the experimental workflow to test the Lactate concentration-dependence of HDAC reversibility using a dialysis system. (H) Representative western blot to assess Kla formation before and after dialysis with different L-Lactate concentrations. This experiment was repeated independently twice with similar results. (I) Schematic of the proposed model of the forward and reverse reactions catalyzed by class I HDACs.