Skip to main content
. 2023 Jun 29;26(7):107235. doi: 10.1016/j.isci.2023.107235

Figure 2.

Figure 2

Absence of H3K9bhb in butyrate-treated HEK293T cells

(A) Schematic of experimental workflow. HEK293T cells were treated with 5 mM BHB or 5 mM butyrate for 24 h. Cell lysates were subjected to immunoprecipitation with α-H3K9bhb antibody or control normal rabbit IgG. The immunoprecipitants were used for Western blot analysis and SDS-PAGE, followed by Coomassie brilliant blue (CBB) staining and LC-MS/MS analysis.

(B) Western blot of input and H3K9bhb IP fractions of HEK293T treated with BHB or butyrate for 24 h. Ponceau S staining was used to confirm the equivalent loading of proteins and H3 enrichment.

(C) Representative tandem mass (MS/MS) spectra of the K9-BHB-ylated and K14-acetylated “PICnKSTGGKAPRR” peptides from BHB-treated samples. The detected y ions and b ions are highlighted in pink.

(D) Left: a strategy of the downstream analysis. Numbers in parentheses refer to the number of detected peptides in BHB or butyrate-treated samples. Right: detected peptide numbers for each modification at the K9 position in the indicated peptides. ND: not determined.