Fig. 4

Missense mutant pVHL constitute E3 ubiquitin complex and retain functionality. a HEK293T cells were transfected with plasmid expressing HA tagged mutant VHL and/or GST tagged Elongin C. At 48 h post-transfection, cells were harvested and lysed in NET gel buffer and then detected the interaction using GST-pull down assay. Interacted mutant pVHLs were detected by immunoblot as indicated. b HEK293T cells were transfected with plasmid expressing GST tagged mutant VHL and/or Flag tagged HIF-1α. At 48 h post-transfection, cells were harvested and lysed in NET gel buffer and then detected the interaction using GST-pull down assay. c HEK293T cells were transfected with HA-tagged mutant VHL and/or Flag-HIF. Cells were incubated for 16 h either in the presence or absence of 10 uM MG132 at 36 h post- transfection. Protein levels are detected by immunoblotting as indicated. d Calculation of levels of HIF-1α at figure C and showed mutant pVHL degraded HIF-1α in cells. e Recombinant missense mutant pVHLs and GST-ODD protein were purified for in vitro ubiquitination assay. Cell lysates from 786-O cell line were provided as supplier of VBC components. His-tagged VHL proteins were incubated with E1, Flag-ubiquitin and GST-ODD protein with cell lystate at 37 °C for 1 h. GST-ODD protein was pulled-down with glutathione sepharose beads and ubiquitinated forms were detected by immunoblot with Flag antibody