Fig. 1: Identification and characterization of β-hydroxybutyrylation (kbhb) modification of p53.

a kbhb modification of overexpressed p53 in 293T cells. Flag-p53 was purified from 293T cells treated with 10 mM β-hydroxybutyrate (BHB), pyruvate or glutamine for 24 h. The purified protein was detected by western blot using anti-pan β-hydroxybutyrylation-lysine antibodies (BHB-K) or anti-p53 (DO-1). b Kbhb modification of endogenous p53 in U2OS cells. Western blot with p53 (DO-1), BHB-K or actin antibodies of cell lysates of U2OS cells treated with 10 mM BHB (bottom half, labeled as “lysate”) or cell lysates immunoprecipitated (IP) with BHB-K antibodies (top, IP: BHB-K) with ReliaBLOT® IP/Western Blot Reagent (Bethyl) to inhibit the signal of the Ig heavy chain interference. c Kbhb modification of endogenous p53 in HCT116 cells. Cell lysates of HCT116 cells treated with or without 10 mM BHB for 24 h were IP with BHB-K antibodies. Western blot analysis of p53 by DO-1 antibodies as in (b). d The BHB in mouse serum were measured. Mice were fasted for 48 h or fed with a normal diet. Blood samples were taken from a tail vein for determination of blood BHB concentrations using a ketone meter. e Kbhb modification of endogenous p53 in mice thymus tissue. Thymus cell lysates of mice fasted for 48 h or fed with a normal diet were IP with BHB-K antibodies. Western blot analysis of p53 with anti-full-length p53 antibodies as in (b). f−h Mass spectrometry analysis identified p53-derived peptides containing β-hydroxybutyrylated Lys-120, Lys-319, and Lys-370. p53 purified from 293T cells transfected with plasmid DNA expressing Flag-p53 and treated with 10 mM BHB for 24 h was analyzed by HPLC-MS/MS. Annotated MS/MS spectra of peptides LGFLHSGTAKSVTCTYSPALMK (derived from amino acids 111−132, f), ALNTSSSPQPKK (derived from amino acids 307−320, g) and AHSSSHLKSK (derived from amino acids 364−372, h) was showing as b- and y-ion coverage. i Effect of lysine-to-arginine mutation on p53 kbhb. Western blot analysis using BHB-K of wild-type and mutant Flag-p53 expressed in 293T cells treated with 10 mM BHB for 24 h