Fig. 5

Crotonylation of GAPDH reduces its catalytic activity. A, B Recombinant GAPDH was incubated with crotonyl-CoA and recombinant p300 and then analyzed by western blotting with the indicated antibodies (A) or assessed for GAPDH activity (NAD⁺ conversion rate) (B). Coomassie blue staining served as loading control. Error bars represent mean ± S.D. (n = 3 independent experiments). Significance was calculated using unpaired t test. **p < 0.01. C H9-LTR7 GFP hESCs were treated with 10 mM crotonate for 12 days and then harvested to assess the enzymatic activity (NAD⁺ conversion rate) of in vivo crotonylated GAPDH. Untreated cells and endodermal cells derived from the reporter cells served as controls. Error bars represent mean ± S.D. (n = 3 independent experiments). Significance was calculated using unpaired t test. **p < 0.01. D LC–MS/MS identified two potential crotonylation sites on GAPDH. Their respective MS/MS spectra are shown as well. E, F Recombinant His-tagged wild-type and mutant GAPDH proteins were purified from E.coli (BL21) and incubated with crotonyl-CoA and recombinant p300. The reaction products were either resolved by SDS-PAGE and probed with the indicated antibodies (E) or assessed for GAPDH activity (NAD⁺ conversion rate) (F). Coomassie blue staining served as loading control. Error bars represent mean ± S.D. (n = 3 independent experiments). Significance was calculated using unpaired t test. *p < 0.05; **p < 0.01. NS, not significant. G, H H9-LTR7 GFP hESCs were cultured with or without 5 μM 3-BrPA for 16 days before being collected for GAPDH activity assessment (NAD⁺ conversion rate) (G) or RT-qPCR analysis of marker gene expression (H). Error bars represent mean ± S.D. (n = 3 independent experiments). Significance was calculated using unpaired t test. **p < 0.01