Fig. 2: Mitochondrial KMT9 regulates PDC activity by monomethylation of DLAT at K596.

a Western blot analysis of PDC immunocaptured from mitochondrial fractions of siCtrl- or siKMT9α-treated PC-3M cells using anti-pan-methyllysine antibody. b Detection of DLAT K596me1 in mitochondrial fractions of DU145 cells treated with siCtrl or siKMT9α and transfected with expression plasmid for LacZ, KMT9α, or catalytically inactive KMT9α (N122A) by Western blot. Membranes were decorated with the indicated antibodies. TOMM20 served as loading control. c In vitro methylation of DLAT. PDC immunocaptured from DU145 cells was incubated with recombinant (r) KMT9α/β or rKMT9α (N122A)/KMT9β and S-adenosyl methionine (SAM). Methylation reactions were analyzed by Western blot using anti-DLAT K596me1 or anti-DLAT antibody. d Detection of DLAT K596me1, KMT9α, and KMT9β in mitochondrial extracts of indicated human specimens by Western blot. e Detection of DLAT K596me1 in nuclear, mitochondrial, or cytosolic fractions of DU145 cells transfected with siCtrl or siKMT9α and expression plasmid for LacZ, MTS-KMT9α, or NLS-KMT9α by Western blot. LMNA, TOMM20, and α-Tubulin served as controls for nuclear, mitochondrial, and cytosolic fractions, respectively. f, g Activity of PDC immunocaptured from extracts of PC-3M cells transfected with expression plasmid for LacZ, DLAT, DLAT (K596R), or DLAT (K547R) in combination with siCtrl or siDLAT. Knockdown efficiency and expression of exogenous DLAT proteins was verified by Western blot (f). Data represent mean + SD from 4 biological replicates. Statistical significance was determined by two-sided Student’s t-test. (a–g) All experiments were independently repeated at least three times with similar results. Source data are provided as a Source Data file.