Fig. 4.

HCV infection and NS3 expression promote the degradation of PPM1A via the ubiquitin proteasome pathway. a Huh-7 cells were infected with JFH1 for 0–5 days as described in Fig. 1a (left panel). The cells were transfected with vector or NS3 for 36 h (right panel), and subsequently, the PPM1A mRNA level was determined by qRT-PCR. Data (n = 3) were normalized to GAPDH and presented as the fold change as compared with control cells. b–c Huh-7 cells were transfected with vector or NS3 for 36 h and subsequently treated with CHX (300 μM) for 0–3 h as indicated (b), vehicle (DMSO), MG132 (20 μM) or chloroquine (50 μM) for 12 h (c). PPM1A protein levels were detected by immunoblotting. d–e Huh-7 cells were infected with JFH1 virus (MOI ~0.5) for 72 h (d), transfected with Flag-vector or Flag-NS3 for 36 h (e), and treated with MG132 (20 μM) for 12 h. The cells were lysed and subjected to immunoprecipitation with antibody against PPM1A and analyzed by western blotting with antibodies against the indicated proteins. Arrows in the anti-ubquitin blots indicate the position of unmodified PPM1A proteins