Figure 2: SIRT7 regulates histone H3K122 succinylation in vivo.

(a) Confirmation of the specificity of H3K122succ (upper) or H2BK120succ (lower) antibodies by peptide competition experiments. Different amounts of soluble histones extracted from U2OS cells were resolved on SDS–polyacrylamide gel electrophoresis (SDS–PAGE) gels, probed with anti-H3K122succ or anti-H2BK120succ with or without excessive H3K122succ or H2BK120succ peptides. The extracted histones were also resolved on SDS–PAGE and stained with Coomassie brilliant blue (CBB). FLAG-H3wt or FLAG-H3K122R was purified from U2OS cells, resolved on SDS–PAGE gels and probed with the anti-H3K122succ antibody alone or anti-H3K122succ antibody pre-adsorbed with H3K122succ peptides or H3K122 control peptides, with H3 analysis as an internal control (middle). (b) SIRT7 or SIRT6 was overexpressed in HEK293T cells or knocked down in MCF-7 cells. Histones were extracted and the succinylation and acetylation were analysed by western blotting with the indicated antibodies. The efficiency of overexpression or KD of SIRT7 and SIRT6 was monitored by western blotting of whole-cell lysate, with corresponding antibodies. (c) MCF-7 cells were treated with 10 μM trichostatin A (TSA), 4 mM of sodium butyrate or 10 mM NAM. Twenty four hours later, cells were collected and soluble histones were prepared and subjected to western blotting with antibodies as indicated.