Immunoprecipitation assay of HIF1α ubiquitination and VHL binding. The 786-0 cells were cotransfected with plasmids expressing pVHL (pCMV6 denotes empty plasmid without VHL open reading frames) and HA-tagged HIF1α. Total protein was extracted from cells and HIF1α was precipitated with magnetic beads coupled with anti-HA antibody. pVHL binding and HIF1α ubiquitination was then tested by western blot. Samples were run on the same gel but were noncontiguous, as indicated by white spaces.