The Salmonella Effector Protein SopA Modulates Innate Immune Responses by Targeting TRIM E3 Ligase Family Members (original) (raw)
Fig 4
TRIM65 interacts with MDA5 and enhances MDA5-stimulated interferon-β expression.
(A) FLAG-epitope-tagged RIG-I (2CARD) (the two CARD domains of RIG-I), RIG-I (full length), MDA5 (2CARD) (the two CARD domains of MDA5), or MDA5 (full length) were transiently expressed in HEK 293T cells together with M45-epitope-tagged TRIM65. Protein interactions were analyzed by immunoprecipitation with anti-FLAG and immunoblotting with anti-M45 and anti-FLAG antibodies. This experiment was repeated 3 independent times with similar results. (B-E) HEK293T cells were co-transfected with plasmids expressing TRIM65 or the catalytically-deficient TRIM65C15A mutant along with plasmids expressing RIG-I (20 ng), MDA5 (50 ng), MDA5(2CARD) (10 ng) or MAVS (50 ng) as indicated, and the reporter plasmids expressing firefly luciferase under the control of interferon-β promoter and renilla luciferase (to standardize the transfection efficiency). The activation of the interferon-β promoter was analyzed 18 h after transfection by Dual Luciferase Reporter Assay System (Promega). Values represent the mean +/- standard deviation of the relative levels of normalized firefly luciferase from 3 independent experiments. The normalized firefly luciferase activity relative to mock-transfected cells was 10.5 ±2.9 for MDA5 only transfected cells (C), 11.1 ± 4.8 for RIG-I only transfected cells (D), 30.6 ± 17.8 for MAVS only transfected cells (E), and 15.4 ± 3.3 for MDA5(2CARD) only transfected cell (F). *: indicates statistically significant difference (p < 0.05) vs. mock transfected cells (B), vs. MDA5-transfected cells (C), vs. RIG-I-transfected cells (D), vs. MAVS-transfected cells (E) and vs. MDA5(2CARD)-transfected cells (F).