Purification and Characterization of Ubiquitin-Conjugating Enzymes

Human WWP1 cDNA (aa2-922) was purchased from Genscript. The ubiquitin variant gene DNA was synthesized by IDT. The WWP1 and ubiquitin variant gene cDNAs were subcloned into a pGEX6p-2 vector for E. coli expression. The WWP1 K740N and N745S mutations were prepared by QuikChange mutagenesis and the sequence of the entire open reading frames were confirmed by DNA sequencing. Wild-type ubiquitin, ubiquitin-activating enzyme UBA1 (E1), and conjugating enzyme UbcH5b (E2) were prepared as previously described^{25 (link)}. The Colloidal Blue staining kit was purchased from Thermo Fisher. The anti-PTEN antibody (N-19 and A2B1) was purchased from Santa Cruz Biotechnology. The anti-GAPDH antibody (14C10) was purchased from Cell signaling Technology. The transfection reagent Lipofectamine 3000 was purchased from Thermo Fisher Scientific. All other reagents were of the highest quality and obtained commercially from either MilliporeSigma or Thermo Fisher Scientific.