Fig 1: AP2M1 and its phosphorylation are important for RABV infection. (A) HEK293 cells were transfected with siRNAs AP2M1 or NT. AP2M1 expression level was determined by Western blotting. ß-actin was used as a loading control. (B) mRNA levels of AP2M1 were determined by qRT-PCR in cells followed by siRNA transfection. A t-test was used for the statistical analysis. ****, p < 0.0001. Representative images of ERA-mCherry-infected (MOI = 0.01) HEK-293 cells (C) and SK cells (E) transfected with siRNAs targeting either AP2M1 or RABV-L, or the NT sequence. A high-content quantitative image-based analysis was used to measure the relative infection ratio (normalized to NT siRNA-treated cells) of RABV in either HEK-293 cells (D) or SK cells (F) transfected with the indicated siRNAs. Cell viability was measured at 48 h post-transfection by using the CellTiter-Glo reagent. Data are relative fluorescence values normalized to the level of the NT control. Values represent the mean ± SD. A one-way ANOVA was used for the statistical analysis. **, p < 0.01, ***, p < 0.001, ****, p < 0.0001; n.s., not significant. (G) HEK-293 cells were treated with various concentrations of sunitinib or erlotinib for 1 h at 37 °C before infection of ERA-mCherry at an MOI of 10, and then incubated for 1 h on ice to allow attachment of viral particles to cell surface receptors. Then the residual viruses were removed, and cell plasma membrane proteins were isolated to detect the phosphorylation of AP2M1 by Western blotting. (H) HEK-293 cells were transfected with pCAGGS-AP2M1(T156A) or pCAGGS. At 48 h post-transfection, the cells were infected with ERA-mCherry (MOI = 0.05), and virus titers were determined as focus forming units (FFU) in BSR-T7/5 cells. Multiple t-tests were used for the statistical analysis, *, p < 0.05. AP2M1-T156 overexpression was confirmed by Western blotting (right panel).