FIG 4.

miR-451a modulates JNK phosphorylation in the context of JEV/WNV infection of neuronal cells. Protein isolated from JEV-infected (MOI 3) Neuro-2A cells (A)/SH-SY5Y cells (B) and WNV-infected (MOI 3) Neuro-2A cells (C) cells for different time-periods as indicated in the figure was investigated for time-dependent expression profile of p-IRE1α, total-IRE1α, p-PERK, total-PERK, ATF-4, CHOP, p-JNK, and total-JNK. Densitometric analysis of immunoblots demonstrate time-dependent kinetics of p-JNK (normalized to total-JNK) in JEV-infected Neuro-2A (A, bottom: ratio of phospho-JNK to total-JNK), JEV-infected SH-SY5Y cells (B, bottom: ratio of phospho-JNK to total-JNK), and WNV-infected Neuro-2A cells (C, bottom: ratio of phospho-JNK to total-JNK). CHOP abundance was evaluated in JEV-infected SH-SY5Y cells (D) or WNV-infected Neuro-2A-cells (E) transfected with miR-451a inhibitor/inhibitor-control or miR-451a-mimic/mimic-control as denoted in the figure. Similarly, p-JNK and total-JNK expression was studied utilizing proteins isolated from JEV-infected (F) and WNV-infected (H) Neuro-2A cells and JEV-infected SH-SY5Y cells (G) transfected with miR-451a inhibitor/inhibitor-control, and miR-451a mimic/mimic-control. All the immunoblots presented in the figure represent data from experimental triplicate with similar results. ß-actin has been used as a loading-control. Molecular weight of each probed molecule has been stated in kDa beside the corresponding immunoblots. Quantitative analysis of immunoblots demonstrating effect of miR-451a inhibitor/inhibitor-control/miR-451a mimic/mimic-control transfection in JEV-infected Neuro-2A (F, bottom: ratio of phospho-JNK to total-JNK), JEV-infected SH-SY5Y (G, bottom: ratio of phospho-JNK to total-JNK), and WNV-infected Neuro-2A (H, bottom: ratio of phospho-JNK to total-JNK) upon p-JNK abundance (normalized to total-JNK). Bar graphs represent data in the form of mean ± SD from three independent experiments with similar outcomes. *, P < 0.05, by two-tailed Student's t test.