The Role of Misshapen NCK-related kinase (MINK), a Novel Ste20 Family Kinase, in the IRES-Mediated Protein Translation of Human Enterovirus 71
Fig 4
Phosphorylation of MINK is triggered post-entry by early replication events.
Phos-tag acrylamide binds phosphorylated proteins and retards their migration to separate the phosphorylated proteins from their unphosphorylated counterparts. Total MINK antibody was used to detect both phosphorylated (upper bands) and unphosphorylated MINK (lower bands). β-actin was used as a loading control. (A) Viral RNA was transfected into cells and cell lysates were harvested at indicated time-points to assess the phospho-MINK levels. Phospho-MINK levels in RNA-transfected cells were comparable to the infection control at the same time-points. (B) The band intensities representing MINK phosphorylation level were quantitated with reference to actin control bands (for each time-point) and 0h using ImageJ Gel Analysis program. (C) Virus titres in the supernatant of cells treated with the anti-SCARB2 and anti-IgG antibodies were analysed via viral plaque assay. Blocking SCARB2 receptors with increasing concentration of SCARB2 antibody resulted in a significant reduction in virus titres. Error bars represent standard deviation (SD) of triplicate data. Statistical analyses were performed using one-way ANOVA and Dunnett’s test (Graphpad software) against untreated control. ***P <0.0001 (n = 3) (D) Blocking SCARB2 receptors with increasing concentration of SCARB2 antibody did not affect the phosphorylation of MINK in cells at 6h after addition of virus. (E) The band intensities representing MINK phosphorylation level were quantitated with reference to actin control bands (for each concentration) and 0μg/mL using ImageJ Gel Analysis program.