(A-B) E7-induced Rb proteolysis does not involve MDM2. (A) C-terminal sequences of Rb are critical for proteolysis by MDM2 not E7. C33A cells were transfected with plasmids expressing V5-epitope tagged ABC-Rb (aa379-928); V5-epitope tagged AB-Rb (379-792) with HPV16 E7 or MDM2. The cell lysates were analyzed for Rb, MDM2 and tubulin (loading control). (B) MDM2 siRNA does not increase the steady state level of Rb in Caski cells. Caski cells were transfected with control siRNA, E7 siRNA, and MDM2 siRNA. After 48h, the cells were harvested and the level of Rb, MDM2, E7, and tubulin (loading control) were analyzed. (C) The catalytic site mutant C92A E2-25K restores the level of Rb in presence of E7. C33A cells were transfected with plasmids expressing V5-ABC-Rb, HPV16 E7, and the active site Cys-Ala mutants of different E2 enzymes as indicated. The cell lysates were analyzed for V5-Rb, E7, and tubulin (loading control).