Fig. 5: USP43 deubiquitinates c-Myc at K148 and K289.
From: USP43 stabilizes c-Myc to promote glycolysis and metastasis in bladder cancer
A 293 T cells were transfected with the indicated siRNAs and plasmids. Cells were treated with 10 μM MG132 for 6 h before harvest, and then ubiquitination experiments were performed to analyze polyubiquitination of c-Myc. B 5637 cells transfected with the indicated siRNA were treated with 10 μM MG132 for 6 h before collection. c-Myc was immunoprecipitated with anti-c-Myc and immunoblotted with anti-ubiquitin. C 293 T cells were transfected with HA-c-Myc, Myc-Ubiquitin and Flag-USP43 (wild-type or C110S). Cells were treated with 10 μM MG132 for 6 h before harvest, and then ubiquitination experiments were performed to analyze polyubiquitination of c-Myc. D Sequence alignment was used to determine the enzyme inactivating mutation site of USP43. E HA-c-Myc was cotransfected with wild-type USP43 or an enzyme inactivating mutant USP43 (C110S) into 293 T cells and subsequent immunoblot analysis. F Schematic diagram of the in vitro ubiquitination experimental procedure. G Deubiquitination of c-Myc in vitro by GFP-USP43. Polyubiquitinated c-Myc was lysed from 293 T cells transfected with HA-c-Myc and Myc-Ubiquitin plasmids and precipitated with anti-HA antibodies. 293 T cells transfected with GFP-USP43 plasmid were lysed and precipitated with anti-GFP antibodies followed by nondenaturing elution to obtain GFP-USP43. Polyubiquitinated c-Myc was incubated with or without GFP-USP43 and then analyzed using IB with anti-Myc antibodies. H 293 T cells were transfected with HA-c-Myc, Myc-Ubiquitin (K48O or K63O) and Flag-USP43. Cells were treated with 10 μM MG132 for 6 h before harvest, and then ubiquitination experiments were performed to analyze polyubiquitination of c-Myc. I HA-tagged wild-type c-Myc and three HA-tagged c-Myc mutants were cotransfected with Flag-USP43 into 293 T cells, and the expression of wild-type c-Myc and c-Myc mutants was detected by an anti-HA antibody. J The effect of USP43 on the ubiquitination level of wild-type c-Myc and three c-Myc mutants was analyzed by deubiquitination assay.
