Fig. 6: CUL5 interacts with and promotes the degradation of O-GlcNAc transferase (OGT).

a Co-immunoprecipitation (Co-IP) and immunoblot analysis of HEK293T cells co-transfected with Flag-OGT and Myc-CUL5. b Co-IP and immunoblot analysis of HEK293T cells co-transfected with Flag-OGT, Myc-CUL5, and Myc-CUL5 mutants including CUL5 ΔCH, CUL5 ΔNEDD, CUL5 ΔCH + ΔNEDD, and CUL5 ΔCR1-3. The protein location is marked by black stars. c Confocal microscopic imaging of HeLa cells co-transfected with Flag-OGT (green), Myc-CUL5, and Myc-CUL5 mutants (red). Nuclei were stained with DAPI (blue). Yellow in merge indicates the co-localization of Myc-CUL5 and Flag-OGT. Scale bars, 10 μm. Stars represent protein size in the indicated group. d Immunoblot analysis of OGT expression in lysates of BMDMs stimulated by 4 μg/ml Poly(I:C) for indicated times. e Immunoblot analysis of the indicated protein expression in lysates of HEK293T cells co-transfected with Flag-OGT and Myc-CUL5. Before collecting cell lysis, cells were pretreated with 10 nM MG132, 200 nM Bafilomycin A1 (BafA1), or 4 uM NH₄Cl. f Immunoblot analysis of OGT ubiquitination in HEK293T cells co-transfected with Flag-OGT, HA-Ub, and CUL5 shRNA. g Immunoblot analysis of OGT ubiquitination in HEK293T cells co-transfected with Flag-OGT, Myc-CUL5, HA-Ub, and Myc-CUL5 ΔCH + ΔNEDD. h Immunoblot analysis of OGT ubiquitination in HEK293T cells co-transfected with Flag-OGT, Myc-CUL5, K48-linked HA-Ub, and the mutant ubiquitin HA-Ub (K48R). i The top six scores of potential ubiquitination sites on OGT predicted according to the BDM-PUB online website. j Immunoblot analysis of OGT ubiquitination with K306R, K459R, K547R, K550R, K996R, and K999R mutations in HEK293T cells co-transfected with HA-Ub, Flag-OGT, and Myc-CUL5. k Structural representation of CRL5 E3 ligases. ELOC, Elongin C; ELOB, Elongin C; SBR, substrate receptor protein; SB, substrates; RBX, RING-box protein; E2, ubiquitin-carrier enzymes; Ub, ubiquitin; NEDD8, Neural precursor cell-expressed developmentally downregulated 8. l Immunoblot analysis of the indicated protein expression in lysates of HEK293T cells co-transfected with Flag-OGT, Myc-CUL5, and Myc-SOCS3. m Co-IP and immunoblot analysis of HEK293T cells co-transfected with Flag-OGT, Myc-CUL5, and Myc-SOCS3. n Immunoblot analysis of OGT ubiquitination in HEK293T cells co-transfected with Flag-OGT, Myc-CUL5, HA-Ub, and Myc-SOCS3. Data are representative of three independent experiments. Source data are provided as a Source Data file.