Figure 6. NS5B-Dependent Interaction between E6AP and pRb.

(A) Endogenous pRb interacts with E6AP in 2–3 replicon cells. Lysates from 2–3 and 2–3c cells were immunoprecipitated with anti-Flag (control), anti-pRb, or anti-MDM2 monoclonal antibody, followed by immunoblotting with anti-pRb and anti-E6AP. All monoclonal antibodies were derived from the same isotype, IgG₁. Ig heavy chain (HC) and nonspecific bands served as loading controls for the pRb and E6AP immunoblots, respectively.

(B) Endogenous pRb interacts with NS5B in 2–3 replicon cells in an EPX-sensitive fashion. 2–3 and 2–3c cells were cultured in the presence or absence of EPX prior to preparation of lysates. Lysates were immunoprecipitated with monoclonal anti-pRb antibody, followed by immunoblotting with anti-pRb and anti-NS5B antibodies. Note that EPX treatment reduces the apparent interaction between pRb and NS5B.

(C) Ectopic expression of wild-type NS5B induces interaction between E6AP and pRb in normal Huh-7 cells. Cells were transfected with vectors expressing Flag-tagged NS3-4A, NS4B, NS5A, NS5B, and a mutant NS5B, D318N/D319N, that does not bind pRb [8], and cultured in the presence of MG115. Cell extracts were immunoprecipitated with anti-pRb antibody, followed by immunoblotting with anti-pRb and anti-E6AP antibodies (top panels). The bottom panels show immunoblots of the cell extracts with the same antibodies.