Prohibitin plays a critical role in Enterovirus 71 neuropathogenesis
(a) Blocking antibody assay. NSC-34 cells were pre-treated with anti-PHB or IgG isotype antibodies, followed by EV71 infection at M.O.I. 10. Culture supernatants were harvested at 48 h.p.i. for viral titer determination by plaque assay. Cell viability was assessed by alamarBlue assay. Statistical analysis was performed using one-way ANOVA with Dunnett’s post-test (***, p<0.0005; ****, p<0.0001). A representative experiment of two independent repeats is shown. (b) Proximity ligation assay. NSC-34 (M.O.I. 20) and RD cells (M.O.I. 5) were incubated with EV71 at 4°C for 2 hours to allow viral adsorption, prior to PLA staining. SCARB2-stained NSC-34 and RD cells served as negative and positive controls, respectively. Scale bar represents 20μm. A representative experiment of two independent repeats is shown. (c) Co-immunoprecipitation of EV71 and surface-expressed PHB. EV71 and NSC-34 cells were co-incubated for 2 hours at 4°C. The cell lysate was pulled down with anti-PHB or IgG isotype control antibodies prior to immunoblotting using anti-VP1 primary antibody. Mock-infected cells and IgG isotype served as control. A representative experiment of two independent repeats is shown. IP, immunoprecipitation; IB, immunoblot.