Figure 6.

SUMOylation-deficient REGγ has reduced activity in p21 degradation. (A) REGγ-6KR influences expression of p21. H1299 cells were transfected with REGγ, REGγ-6KR, an empty vector, or a mock control along with p21 for 24 h. Expression of the protein of interest was examined by western blotting with indicated antibodies. CDK9 serves as a control for specificity. (B) SUMOylation-defective REGγ has attenuated proteolytic activity. Experiments were performed as in (A) and cells were treated with cycloheximide for the indicated time. The expression of p21 and dynamic decay rate were determined by western blot analysis. The expression of REGγ and REGγ-6KR was equivalent in the two groups of samples examined. (C) SUMOylation-deficient REGγ-6KR has attenuated affinity for p21. In vivo interaction between p21 and wt or SUMOylation-defective REGγ was examined by co-expressing these constructs and by subsequent immunoprecipitation analysis using anti-Flag antibody. Expression of a Flag-empty vector was used as negative control. To prevent degradation of the substrate p21, cells were treated with MG132 for 4 h before collection.