Figure 5

miR-146a upregulation by JEV targets STAT1 phosphorylation. (A) CHME3 cells were infected with JEV and pelleted at 12 and 24 h post infection. Western blots representing phospho-STAT1 and STAT1 levels at 12 and 24 h post infection. Both STAT1 and phospho-STAT1 got downregulated 24 h post infection. (B) Densitometry plot showing increase in phosphorylation of STAT1 at 12 h post infection. Later, no increase in phosphorylation was observed 24 h after infection due to downregulation in levels of STAT1. Both phospho-STAT1 and STAT1 image density was normalized by β-tubulin. For statistical analysis, the total and phospho-STAT1 levels of 12 and 24 h JEV-infected samples were compared to control uninfected samples at 12 and 24 h. (C) Western blot showing downregulation of STAT1 upon miR-146a mimic overexpression. (D) Western blot showing that silencing of miR-146a by anti-miR-146a upregulates STAT1 upon JEV infection. (E) Densitometry plot showing upregulation of STAT1 upon silencing of miR-146a. JEV infection also upregulates STAT1 when anti-miR-146a is transfected 24 h prior to JEV infection. The image density was normalized by β-tubulin. For statistical analysis, the anti-miR-146a and anti-miR-146a + JEV groups were compared to JEV-infected group. All experiments were repeated thrice and are represented as mean ± SE. The fold change is significant where *denotes P < 0.05, **denotes P < 0.005, and ***denotes P < 0.001.