FIG 2.

Regulation of E6AP through T485 affects degradation of the E6AP's normal substrate, Ring1B. (A) E6AP-null HEK293 cells were transfected with the indicated plasmids, and after 24 h the cells were harvested and protein levels analyzed by Western blotting (WB). β-Galactosidase (β-Gal) acted as a control for transfection efficiency. WT, wild type. (B) Cells were transfected with the indicated expression plasmids, and after 24 h they were harvested and subjected to immunoprecipitation (IP) with anti-FLAG-conjugated agarose beads. Polyubiquitinated Ring1B was then detected by Western blotting with anti-HA-HRP antibody. (Lower) Input levels of Ring1B used in the immunoprecipitation.