Fig. 4.
A, detection of SUMOylated human PXR protein in HeLa cells. The human PXR protein was coexpressed in HeLa cells together with either His-tagged SUMO1, SUMO2, and SUMO3 proteins. Cells were lysed using denaturing buffer containing guanidinium hydrochloride to inactivate de-SUMOylation enzymes. SUMOylated proteins were purified by using nickel-linked agarose beads. The blot was probed for PXR immunoreactivity using a monoclonal antibody that recognizes the human PXR protein. B, dominant-negative Ubc9 (C93S) protein inhibits SUMOylation of human PXR in a dose-dependent manner. The human PXR protein was coexpressed in HeLa cells together with 6×His-tagged SUMO3 and increasing amounts of dominant-negative Ubc9 (C93S). SUMOylated protein was purified using nickel-linked agarose beads. Left, the blot was probed for PXR immunoreactivity using a monoclonal antibody that recognizes the human PXR protein. Right, the blot was stripped and reprobed with an antibody that recognizes SUMO2/3.