FIGURE 1

RBCK1 regulates SeV-triggered type I IFN signaling. (A) RBCK1 inhibits SeV-induced activation of IFN and IFN4 promoters. The 293 cells (510⁴) were transfected with IFN or IFN4 promoter luciferase plasmid (50 ng), pRL-TK Renilla luciferase plasmid (50 ng), and an expression vector for Flag-RBCK1 (0.1 g) or an empty vector (0.1 g). At 12 h after transfection, cells were infected with SeV (filled bars) or left uninfected (open bars) for 12 h before luciferase assays were performed. (B) RBCK1 inhibits SeV-induced expression of endogenous IFN and IFN4. The 293 cells (210⁵) were transfected with an expression vector for Flag-RBCK1 (0.5 g) or an empty vector (0.5 g). At 12 h after transfection, cells were infected with SeV or left uninfected for 12 h before RT-PCR analysis of the indicated mRNAs. (C) Effects of RBCK1 RNAi plasmids on the expression of overexpressed and endogenous RBCK1. The 293 cells (210⁵) were transfected with HA-tagged RBCK1 and Dynactin (upper panels) or empty control plasmid (lower panels) (1 g each), together with pSuper control, RBCK1-RNAi-#1 or RBCK1-RNAi-#4 plasmid (2 g each) as indicated. At 48 h after transfection, cell lysates were analyzed by Western blot with anti-HA (upper panels), anti-RBCK1, or anti-GAPDH (lower panels) antibody. (D) Knockdown of RBCK1 potentiates SeV-triggered activation of IFN and IFN4 promoters in 293 cells. The 293 cells (510⁴) were transfected with the IFN or IFN4 promoter reporter plasmids (50 ng) and the indicated RBCK1 RNAi plasmid (1 g). At 36 h after transfection, cells were infected with SeV or left uninfected for 12 h before luciferase assays. (E) knockdown of RBCK1 increased SeV-triggered expression of endogenous IFN and IFN4. The 293 cells (210⁵) were transfected with a control or the indicated RBCK1 RNAi plasmid (2 g each). Thirty-six hours after transfection, cells were infected with SeV or left uninfected for 12 h before RT-PCR analysis of the expression of the indicated mRNAs.