FIG 5.

VDAC1 enhances IBDV polymerase activity in vitro. (A and B) Polymerase activity assays were performed with 293T cells expressing an IBDV segment B-driven luciferase minigenome system including segment A, segment B, TK, and VDAC1 or empty vector. VDAC1, VP1, and VP3 protein expression was analyzed by Western blotting. (C and D) Polymerase activity assays were performed as described for panels A and B, but with VP3 instead of segment A. VDAC1, VP1, and VP3 protein expression was analyzed by Western blotting. Endogenous β-actin expression was used as an internal control. (E) Effects of VDAC1 RNAi on the expression of endogenous VDAC1. 293T cells were transfected with specific siRNA (RNAi#1, RNAi#2, or RNAi#3) or a control. Cell lysates were harvested 48 h after the second transfection and examined by Western blotting with anti-VDAC1 antibody. Endogenous β-actin expression was used as an internal control. (F) Relative intensities of VDAC1 in VDAC1 siRNA-treated cells. β-Actin was used as a protein loading control. Representative results are shown, with bars representing the intensities of VDAC1/actin bands normalized to the control condition. (G and H) 293T cells were transfected with scrambled control or VDAC1 siRNA. Polymerase activity assays were performed 36 h after transfection as described for panels C and D. VDAC1, VP1, and VP3 protein expression was analyzed by Western blotting. Endogenous β-actin expression was used as an internal control.