TRIM38 Negatively Regulates TLR3-Mediated IFN-β Signaling by Targeting TRIF for Degradation
Figure 2
Effects of TRIM38 knockdown on TLR3-induced IFN-β activation.
(A) TRIM38 protein level in 293/TLR3 cell line stably expressing TRIM38-specific or non-targeting shRNA. 293/TLR3 cells were transfected with TRIM38-specific shRNA or non-targeting shRNA (NT) followed by puromycin selection and cloning. Screened cell populations were analyzed by immunoblot with an antibody specific for TRIM38. (B) Effects of TRIM38 knockdown on poly(I:C)-induced IFN-β activation. TRIM38 knockdown or control 293/TLR3 cells were transfected with IFN-β-luc plasmid. Twenty-four hours after transfection, cells were left untreated or stimulated with 100 µg/ml of poly(I:C) for 4 h before luciferase analysis was performed. (C) Effects of TRIM38 knockdown on poly(I:C)-induced IRF3 phosphorylation. TRIM38 knockdown or control 293/TLR3 cells were incubated with 100 µg/ml of poly(I:C) for 4 h before the immunoblot analysis was performed. (D, E) Effects of TRIM38 knockdown on poly(I:C)-induced transcription of IFN-β and ISG56 genes. Indicated cells were stimulated with poly(I:C) for 4 h, then total RNA was extracted for real-time PCR analysis. Similar results were obtained from three independent experiments.