Knockdown of GPATCH3 enhances the activation of RLR-mediated signaling.
(A) 293T cells (4 x 105) were cotransfected with Flag-GPATCH3, HA-actin (0.1 μg each) and control- or the indicated GPATCH3-shRNA plasmids (1 μg each). Twenty-four hours after transfection, cell lysates were analyzed by immunoblotting with anti-Flag or anti-HA antibodies (upper panel). 293T cells (4 x 105) were transfected with control- or the indicated GPATCH3-shRNA plasmids (1 μg each). Thirty-six hours after transfection, cell lysates were analyzed by immunoblotting with anti-GPATCH3 or anti-β-actin antibodies (lower panel). (B) 293T cells (1 x 105) were transfected with control- or the indicated GPATCH3-shRNA plasmids (0.25 μg each) together with the indicated reporters (0.05 μg each). Thirty-six hours later, cells were left uninfected or infected with SeV for 12 hours before luciferase assays were performed. (C) 293T cells (4 x 105) were transfected with control- or GPATCH3-shRNA plasmids (1 μg each). Thirty-six hours later, cells were left uninfected or infected with SeV for 12 hours before total RNAs were extracted and the mRNA levels of the indicated genes were analyzed by qPCR. (D) 293T cells (4 x 105) were transfected with control- or GPATCH3-shRNA plasmids (1 μg each). Thirty-six hours later, cells were left uninfected or infected with SeV for the indicated times. Cell lysates were then analyzed by immunoblotting with the indicated antibodies. (E&F) A549 cells (4 x 105), HFFs (4 x 105) and PBMCs (1 x 106) were transfected with control or GPATCH3 siRNAs (40 nM). Thirty-six hours later, cells were left untreated or treated with the indicated infections or transfections. Twelve hours later, total RNAs were extracted and the mRNA levels of the indicated genes were analyzed by qPCR. Graphs show mean ± SD. n = 3. *P<0.05, **P<0.01 (Student’s t-test).
