Fig 4. EV71 3Cpro cleaves Ubc6e at multiple distinct sites.
(A) 293T cells were transfected with increasing doses of plasmids (0–4 μg) encoding GFP-3C or GFP-3C(C147S). At 36 h post-transfection, cells lysates were analyzed by western blotting with antibodies against Ubc6e (mouse monoclonal antibody) and GFP. Arrows indicate the cleavage fragments (CF). (B) 293T cells lysates were incubated with 100 or 200 ng/μl recombinant 3Cpro or 3C protease-dead mutant 3Cpro(E71A) at 30°C for 2 h, then the mixture was subjected to western blot analysis with indicated antibodies. (C) RD cells stably expressing SHH-FLAG were transfected with plasmid encoding GFP or increasing doses of plasmid encoding 3C-FLAG (0–1.5 μg). At 36 h after transfection, the cells were treated with (+) or without (−) CHX for 4 h. Mock- and EV71-infected cells were used as the negative and positive controls for Ubc6e cleavage, respectively. Cells lysates were analyzed by western blotting with antibodies against Flag, Ubc6e, EV71 3C, and Actin. Arrows indicate the cleavage fragments (CF) of Ubc6e. The lower panel graph shows the quantification of SHH-C. The data are presented as means ± SD of two independent experiments. (D) 293T cells were transfected with plasmid encoding Ubc6e alone or together with plasmid encoding GFP-3C. At 36 h after transfection, cells lysates were analyzed by western blotting with Ubc6e (left panel) and FLAG (right panel) antibodies. (E) Schematic diagram of the Ubc6e amino acid sequence and structural domains (** represents the identified 3Cpro cleavage sites on Ubc6e). (F–H) 293T cells were transfected with plasmids encoding Ubc6e/Ubc6e mutants with or without plasmid encoding GFP-3C. At 36 h after transfection, western blotting was performed to detect Ubc6e cleavage using Ubc6e and GFP antibodies. Arrows indicate cleavage fragments (CF) and asterisks indicate the phosphorylated form of cleavage bands. The numbers on the lane indicate the cleavage sites of cleavage fragments. (I) 293T cells were transfected with plasmid encoding Ubc6e alone or together with plasmid encoding GFP-3C. At 36 h after transfection, cells were lysed and cell lysates were incubated with (+) or without (−) Lambda protein phosphatase at 30°C for 30 min, then the lysates were analyzed by western blotting with antibodies against Ubc6e, GFP, and actin.