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. Author manuscript; available in PMC: 2024 Nov 1.
Published in final edited form as: Nat Chem Biol. 2023 Aug 31;19(11):1423–1431. doi: 10.1038/s41589-023-01422-2

Extended Data Figure 5. The Int-D binding site is a motif-dependent regulator of protein association and O-GlcNAcylation.

Extended Data Figure 5.

Co-immunoprecipitation of cMyc-SMG9 with Flag-OGT (WT) or (a) Flag-OGT-I734R (I734R), (b) Flag-OGT-I787E (I787E), (c) Flag-OGT-I787E-N791A (2M), from TRex-293 cells, followed by western blot. (d) Co-immunoprecipitation of endogenous OGA with Flag-OGT (W) or Flag-OGT-N791A (M) from TRex-293 cells, followed by western blot. (e) O-GlcNAcylation detection on CaMKMT from cells co-expressed with Flag-OGT (W) or Flag-OGT-N791A (M). HA-tagged CaMKMT was immunoprecipitated from TRex-293 cell lysate by anti-HA agarose and the O-GlcNAcylation was detected with the same GalT assay as SMG9. (f) O-GlcNAcylation detection on Flag-OGT (W), Flag-OGT-N791A (M), and Flag-HA-OGA-D175N. Flag-tagged OGT and OGA were immunoprecipitated from TRex-293 cell lysate by Flag-agarose, followed by western blot. All whole cell lysates have endogenous OGT knocked down. Blots are representative of at least three biological replicates.