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. 2021 Aug 24;13(9):1674. doi: 10.3390/v13091674

Figure 3.

Figure 3

THGP interacts with 3pRNA but not Poly I:C and HT-DNA and inhibits the binding of RIG-I with 3pRNA. (A,B) Luciferase assay of IFN-β gene promoter after treatment of the indicated concentrations of THGP following the transfection of 3pRNA (A), RIG-I (B), and MAVS (C) in HEK293T cells. (D) FACS analysis at 2 h after transfection with Cy5-3pRNA in RAW264.7 cells. (E) THGP beads pull down assay of 3pRNA, poly I:C, and HT-DNA. The amount of precipitated RNA/DNA with THGP beads (filled bar) or control beads (opened bar) is shown. (F) THGP beads pull down assay of 3pRNA and 3pRNA treated with alkaline phosphatase. The amount of precipitated RNA/DNA with THGP beads (filled bar) or control beads (opened bar) is shown. (G) Pull-down assay showing the binding of biotinylated 3pRNA to RIG-I (top) and THGP to RIG-I (bottom). (H,I) 3pRNA (H) and polyI:C (I) pull down assay, which test the interaction of biotinylated 3pRNA and RIG-I and biotinylated polyI:C and MDA-5 in RAW264.7 cells. Co-precipitated proteins with biotinylated RNA in the presence of indicated concentrations of THGP were subjected to Western blotting using anti-RIG-I and anti-MDA-5 antibodies. (J) Immunofluorescence analysis for the co-localization of RIG-I and Cy5-3pRNA in the presence of the indicated concentrations of THGP. A representative of more than 30 captured cells is presented. Bar: 10 μm. ** p < 0.01 vs. control. Data are presented as mean and s.d. (n = 3) and are representative of at least three independent experiments.