Fig. 2

UCP interacted missense mutant pVHLs directly and ubiquitinated in vitro and in cell. a GST tagged recombinant proteins of mutant pVHLs were ubiquitinated by UCP in vitro. GST-pVHL (V155A, L158Q, Q164R) and/or His-UCP protein were incubated at 37 °C in the presence of E1 and Flag-ubiquitin. GST-pVHL polyubiquitination was detected by western blot with Flag antibody. b Each mutant GST-VHL protein and His-UCP protein were mixed and then detected the interaction using GST-pull down assay. Bound UCP protein was detected by immunoblot. c Plasmid expressing GST tagged mutant VHL were transfected into HEK293T in the presence of MG132 for 16 h and then ubiquitinated forms were isolated using GST resin and then detected by immunoblot using antibody to detect HA-Ub conjugated pVHL. d Plasmid expressing GST tagged mutant VHL and /or Flag-UCP were transfected into HEK293T in the presence of MG132 for 16 h and then mixed and then detected the interaction using GST-pull down assay. Bound UCP protein was detected by immunoblot