Extended Data Fig. 10: Stoichiometry of K108bhb in mice livers or in cells.
From: Ketogenic diet reshapes cancer metabolism through lysine β-hydroxybutyrylation
a, b Stoichiometry of K108bhb in mouse livers. The liver samples contained same amount of Aldob as purified Aldob-K108bhb protein. Aldob-K108bhb protein was diluted at different fold, loaded to a gel together with the liver samples, and probed using Aldob K108bhb-specific antibody. The stoichiometry of K108bhb was determined by comparing densitometry of the liver samples with that of purified protein. c Lysosome fractions were isolated and probed by immunoblotting with different marker antibodies. d, e The lysosomal samples contained same amount of Aldob as purified Aldob-K108bhb protein. Aldob-K108bhb protein was diluted at different fold, loaded to a gel together with the lysosome samples, and probed using Aldob K108bhb-specific antibody. The stoichiometry of K108bhb was determined by comparing densitometry of the lysosomal samples with that of purified protein. p value: 5.2e − 5; f, g Stoichiometry of K108bhb in HCC-LM3 cells expressing Flag-Aldob-K147R. Aldob-K147R was immunoprecipitated by the Flag tag, and same amount of Aldob-K147R was used as purified Aldob-K108bhb protein. Aldob-K108bhb protein was diluted at different fold, loaded to a gel together with Aldob-K147R, and probed using Aldob K108bhb-specific antibody as described above. h Immunoblotting and coomassie brilliant blue staining analysis of purified Aldob WT and K108ac. i Intact mass measurement by MALDI-TOF verified the successful introduction of protein acetylation at K108 of Aldob. Calculated mass: 43162. j, k Stoichiometry of Kac in HCC-LM3 cells expressing Flag-Aldob-K147R. Aldob-K147R was immunoprecipitated by the Flag tag, and same amount of Aldob-K147R was used as purified Aldob-K108ac protein. Aldob-K108ac protein was diluted at different fold, loaded to a gel together with Aldob-K147R, and probed using Kac antibody. Similar results were obtained in three independent experiments (a-h, j-k). Statistical data are presented as mean ± SEM (b, e, g, k), P values were determined by unpaired two-tailed t test (b, e, g, k).
