Fig. 4

Large-scale analysis of lysine-crotonylated proteins in crotonate-treated hESCs identifies glycolytic enzymes as major targets. A Reporter hESCs were cultured in crotonate-containing media (10 mM) for 12 days before being harvested along with untreated hESCs for protein extraction and peptic digest. Crotonylated peptides were enriched with an anti-Kcr antibody before LC/MS–MS analysis. B GO analysis of crotonylated proteins in response to crotonate treatment that were identified by LC/MS–MS. C H9-LTR7 GFP reporter hESCs and endodermal cells derived from the reporter cells were immunoprecipitated (IP) with an anti-GAPDH antibody. The immuoprecipitates were then western blotted as indicated. IgG served as a negative control. D Reporter hESCs stably expressing HA-tagged GAPDH (left) or ENOA (right) were cultured in crotonate-containing media (10 mM) for 24 h before being harvested for IP with the anti-Kcr antibody and western blotting with an anti-HA antibody. Parental (Mock) and untreated cells served as controls