a, c, e, Extracted ion chromatograms from HPLC–MS/MS analysis of histone Kla peptides derived from cultured cells (in vivo), the synthetic counterparts, and their mixtures. b, d, MS/MS spectra of histone Kla peptides derived from in vivo, the synthetic counterparts, and their mixtures. f, g, Antibody specificity tests by dot blot and competition assay. f, Dot blot was carried out with a pan anti-Kla antibody and the following peptide libraries. A1, A2, A3 and A4: dots contain 1, 4, 16 and 64 ng, respectively, of a peptide library containing a lactylated lysine residue. B1, B2, B3 and B4: dots contain 64 ng of a peptide library containing an unmodified (K), acetylated (Kac), propionylated (Kpr) and butyrylated (Kbu) lysine residue, respectively. C1, C2, C3 and C4: dots contain 64 ng of a peptide library containing a β-hydroxybutyrylated (Kbhb), 2-hydroxyisobutyrylated (Khib), crotonylated (Kcr) and malonylated (Kma) lysine residue, respectively. The libraries contained a mixture of CXXXKXXXX peptides, in which C is cysteine, X is a mixture of all 19 amino acids except for cysteine, and K is lysine with or without the indicated modifications. g, Competition was carried out by incubating the pan anti-Kla antibody with a twofold or tenfold excess of the indicated peptide libraries before the dot blot assay. h–j, Exogenous lactate boosts histone Kla levels. Immunoblot analysis of histone Kla and Kac from human MCF-7 cells treated with indicated doses of l-lactate (h), and from human HeLa (i) and MDA-MB-231 (j) cells treated with 25 mM sodium chloride, sodium lactate or sodium acetate. k, MS/MS spectra of an isotopically labelled histone Kla peptide identified from MCF-7 cells cultured with 10 mM isotopic (13C 3 ) sodium l-lactate for 24 h. Data in a–k represent three independent experiments.