Fig. 6: FBXO31 interacts with and ubiquitinates OGT. | Nature Communications

Fig. 6: FBXO31 interacts with and ubiquitinates OGT.

From: FBXO31-mediated ubiquitination of OGT maintains O-GlcNAcylation homeostasis to restrain endometrial malignancy

Fig. 6: FBXO31 interacts with and ubiquitinates OGT.The alternative text for this image may have been generated using AI.

a Immobilized recombinant GST-OGT protein but not GST control absorbed GFP-FBXO31 from 293T cell lysates. GST and GST-OGT were detected by Coomassie brilliant blue (CBB) staining, and FBXO31 was detected by western blotting with FBXO31 antibody. b Co-immunoprecipitation of GFP-FBXO31 with Flag-OGT in 293T cell lysates. The presence of MG132 enhanced the interaction between Flag-OGT and GFP-FBXO31. c Western blotting assessing the protein level of OGT as well as the global O-GlcNAcylation (RL2) level in 293T cells transfected with increasing amount of GFP-FBXO31. d Western blotting quantification of the protein level of endogenous OGT in 293T cells transfected with GFP-FBXO31. MG132 was added to inhibit the ubiquitination-mediated proteasome degradation. e Western blotting detecting the protein level of endogenous OGT and its ubiquitination in 293T cells transfected with different amount of HA-Ub and GFP-FBXO31. f In vitro ubiquitination of His-OGT by the SCF complex together with FBXO31. HA-tagged SCF components (Skp1, Cul1, and Roc1) and HA-FBXO31 were affinity-purified using anti-HA-conjugated magnetic beads from 293T cell lysates. The purified protein complex was incubated with E1 (UBA1), E2 (UBE2D1), Ub, and His-OGT in ubiquitination buffer. The reaction was halted by the addition of SDS sample buffer, and the samples were subjected to western blotting using the indicated antibodies. g In vivo ubiquitination assay was performed to evaluate the ubiquitination levels of exogenous Flag-OGT in 293T cells transfected with HA-tagged Ub and GFP-FBXO31 or its F-box domain deletion mutant GFP-FBXO31ΔF. h Western blotting detecting the O-GlcNAcylation (RL2) and OGT levels in WT and FBXO31-KO 293T cells. i Western blotting quantitation of OGT protein level following cycloheximide (CHX) treatment in WT and FBXO31-KO 293T cells. Results in (d, i) represent n = 3 independent experiments, with p-values calculated by unpaired two-tailed Student’s t-test and data presented as mean ± SD. Samples derive from the same experiment and gels were processed in parallel. (a–c, e–h) show representative examples from n = 3 independent experiments. The source data for results in (a–i) are provided in the Source Data file.

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