Fig. 1: XIAP acts as an E3 ubiquitin ligase for OGT.

a HCT16 cell lysates were incubated with immobilized recombinant GST-OGT fusion protein (residues 1–1036). GST-OGT was precipitated and the associated XIAP was detected by western blotting with α-XIAP antibodies. GST and GST-OGT were detected by Coomassie brilliant blue staining to verify the amount of protein used in the assay. b HCT116 cells transiently overexpressing Flag-tagged XIAP were immunoprecipitated with α-Flag or IgG control antibodies. Co-immunoprecipitated endogenous OGT was detected by α-OGT antibodies. The same membrane was re-probed with α-XIAP antibodies. Total lysates were blotted with α-OGT and α-XIAP antibodies. c HCT116 cells were transfected with Flag-tagged OGT and immunoprecipitated with α-Flag or IgG control antibodies. Bound endogenous XIAP was detected by α-XIAP antibodies. The same membrane was re-probed with α-OGT antibodies. Total lysates were blotted with α-OGT and α-XIAP antibodies. d OGT in vivo ubiquitination assay. Expression vectors encoding Flag-OGT and HA-ubiquitin (Ub) were transfected into HCT116 cells transiently overexpressing Myc-XIAP as indicated. The cells were treated with 20 μM of MG132 for 4 hours (h) before they were harvested. After being immunoprecipitated with α-Flag antibodies, the ubiquitination of OGT was analyzed by immunoblotting with α-HA antibodies. The same membrane was re-probed with α-Flag antibodies. Equal amounts of total lysates were subjected to immunoblotting with the indicated antibodies. e OGT in vitro ubiquitination assay. GST-OGT was incubated with His-XIAP, HA-Ub, E1, E2, and ATP as indicated. After GST pull-down under denaturing conditions with a buffer containing 2% SDS, the ubiquitination of OGT was analyzed by immunoblotting with α-HA antibodies. The same membrane was re-probed with α-GST antibodies. Equal amounts of XIAP in the reaction were immunoblotted with α-His antibodies. f HCT116 WT or HCT116 XIAP KO cells were transfected with Flag-OGT and treated with 20 μM of MG132 for 4 h. The ubiquitination of immunoprecipitated OGT was analyzed by immunoblotting with α-Ub antibodies. The same membrane was re-probed with α-Flag antibodies. Equal amounts of total lysates were subjected to immunoblotting as indicated. β-actin was used as a loading control. All data are representative of at least three independent experiments.