Fig. 4.

ESRRB O-GlcNAcylation enhances protein stability, protein-protein interactions and transcriptional activity. a Immunoblots showing expedited degradation of ESRRB upon O-GlcNAcylation inhibition. mESCs expressing FLAG-ESRRB pre-treated with or without Ac₄5SGlcNAc were incubated with 50 μM CHX for up to 8 h, during which the expression of FLAG-ESRRB was monitored. b Immunoblots showing mESCs expressing FLAG-ESRRB or FLAG-ESRRB^S25A treated with CHX for up to 12 h. c Immunoblots showing HEK293T cells expressing EGFP-ESRRB or EGFP-ESRRB^S25A treated with CHX for 12 h. Bar graph showing statistical analysis of the relative protein level of HEK293T cells treated with CHX for 12 h (normalized to that with no CHX). Error bars represent mean ± s.d. ***P < 0.001 (Student’s t-test). d Immunoblots showing co-immunoprecipitation of endogenous OCT4 and NANOG with ESRRB in mESCs. mESCs expressing FLAG-ESRRB or FLAG-ESRRB^S25A were immunoprecipitated with an anti-FLAG antibody. e, f Immunoblots showing co-immunoprecipitation of OCT4 and NANOG with ESRRB in HEK293T cells. The cells co-expressing FLAG-OCT4 with EGFP-ESRRB or EGFP-ESRRB^S25A or co-expressing FLAG-NANOG with EGFP-ESRRB or EGFP-ESRRB^S25A were immunoprecipitated with an anti-GFP antibody. g, h Increasing O-GlcNAcylation promoted interactions of OCT4 and NANOG with ESRRB. With the treatment of DMSO or Thiamet-G (TG) for 48 h, HEK293T cells co-expressing FLAG-OCT4 with EGFP-ESRRB or co-expressing FLAG-NANOG with EGFP-ESRRB were immunoprecipitated with an anti-GFP antibody. i Luciferase assay showing reduced ESRRB transcriptional activity upon O-GlcNAc inhibition. j Luciferase assay showing transcriptional activities of WT and S25A mutant ESRRB. In i and j results are shown from three independent experiments. ***P < 0.001 (one-way ANOVA). The anti-GAPDH gels in a–f demonstrate comparable loading. In a–h representative results are shown from three independent experiments. Source data for figures a–j are provided as a Source Data file