Figure 6
From: Foot-and-mouth disease virus non-structural protein 3A inhibits the interferon-β signaling pathway
FMDV 3A inhibits the expression of the RIG-I, MDA5 and VISA.
(a–d) The effects of FMDV 3A on the expression of the RIG-I, MDA5 and VISA. HEK293T (1 × 105) cells were co-transfected with increased amounts of pCAGGS-Flag-3A (F-3A) (0, 0.5, 1.0 and 2 μg) and decreased amounts of pCAGGS (2.0, 1, 1.5 and 0 μg) and indicated plasmids expressing pRK-HA-RIG-I, pRK-HA-MDA5, pRK-HA-VISA or pRK-HA-TBK1 (0.2 μg). As a control, pRK-HA-actin (0.015 μg) was transfected with above plasmids. At 48 hour post transfection, the cell lysate was analyzed by Western blotting using mouse anti-Flag and mouse anti-HA monoclonal antibodies. HA-actin was used as a loading control. (e) Dose-dependent effects of FMDV 3A on the expression of endogenous RIG-I, MDA5, VISA, TBK1, TRAF3 and IRF3. HEK293T cells (1.6 × 107) were co-transfected with increased amounts of pCAGGS-Flag-3A (F-3A) (0, 4, 8.0, 12, 16, 24 and 32 μg) and decreased amounts of pCAGGS (32, 28, 24, 20, 16, 8 and 0 μg) for 24 h. A small volume of the protein lysate was collected to detected VISA, TBK1, TRAF3 and IRF3 by western blotting. Other protein lysate was used for immunoprecipitation with protein G-beads coupled to polyclonal antibodies directed against RIG-I or MDA5. The immunoprecipitation was analyzed by immunoblots with the polyclonal antibodies against RIG-I and MDA5.
