Fig. 7: Cdc42 acts as a molecular switch regulating c-Myc degradation by WWP2.

A The ubiquitination level of c-Myc in GSC07 cells treated with or without ML141 (10 μmol/l) for 24 h. B, C c-Myc level in T98G cells transfected with GFP-Tagged Cdc42^WT, Cdc42^T17N or Cdc42^Q61L. D Schematic diagram depicting the plasmid construction for bimolecular fluorescence complementation assays. E Schematic illustration of the bimolecular fluorescence complementation assay used to assess the interaction between Cdc42 and WWP2. F HEK-293 cells were transfected with CrN173-tagged Cdc42^T17N or Cdc42^Q61L, and VC155-tagged WWP2 for 48 h. CFP signal that represents Cdc42 binding to WWP2 were determined using confocal. Scale bar, 5 μm. G HEK-293 cells were transfected with Flag-tagged c-Myc, HA-tagged ubiquitin, MYC-tagged WWP2 and GFP-tagged Cdc42^T17N or Cdc42^Q61L for 48 h. The ubiquitin level of c-Myc and the binding of Cdc42 to WWP2 were determined by co-immunoprecipitation. H Schematic illustration of the bimolecular fluorescence complementation assay used to assess the interaction between WWP2 and Cdc42 or c-Myc. I HEK-293 cells were incubated with or without SEMA3G (200 ng/ml) for 48 h after co-transfected with CrN173-tagged Cdc42^WT or VN173-tagged c-Myc and VC155-tagged WWP2 for 12 h. CFP signal that represents Cdc42 binding to WWP2 and Venus signal that represents WWP2 binding to c-Myc were captured using confocal. Scale bar, 5 μm. J HEK-293 cells that were co-transfected with GFP-tagged Cdc42^WT, MYC-tagged WWP2, Flag-tagged c-Myc, and HA-tagged ubiquitin (UB) for 12 h, followed by the treatment with or without SEMA3G (200 ng/ml) for 48 h. The binding of Cdc42^WT to WWP2 and the ubiquitination level of c-Myc were determined by co-immunoprecipitation. K HEK-293 cells were co-transfected with GFP-tagged Cdc42^Q61L and MYC-tagged WWP2, Flag-tagged c-MYC, and HA-tagged ubiquitin (UB) for 12 h, followed by the treatment with or without SEMA3G (200 ng/ml) for 48 h. The protein level of c-Myc and ubiquitination level of c-Myc were determined by co-immunoprecipitation. L Schematic diagram illustrating the potential mechanism by which Cdc42 regulates WWP2 binding to c-Myc and promotes c-Myc degradation. All the western blot bands represent one of the three independent experiments.