Figure 9.

Function of different Danio rerio DDX41 (DrDDX41) domains in the signaling pathways. (A) Schematic diagram of full-length and mutated DrDDX41 fragments, which were inserted into the C-terminus of pCMV-Tag2B. (B,C) DrDDX41 (B) and its mutants [(C), constructed as in panel (A)] interact with Danio rerio STING (DrSTING). HEK293T cells were transfected with Flag-DrDDX41/its mutants and Myc-DrSTING for 24 h. Cell lysates were immunoprecipitated with anti-Myc antibody (Myc) and analyzed by western blot using anti-Flag and anti-Myc antibodies. Expression of the transfected plasmids was analyzed with anti-Flag and anti-Myc antibodies in the whole cell lysates. (D–F) Activation of the nuclear factor-κB (NF-κB)-binding (D), HsIRF3 (E), or HsIFNβ (F) promoters in HEK293T cells transfected with an NF-κB/HsIRF3/HsIFNβ-Luc reporter (150 ng/mL) and a renilla luciferase reporter (15 ng/mL) plus different vectors of DrDDX41 (400 ng/mL) together with pCMV-N-Myc-DrSTING (200 ng/mL) and stimulated with poly(dA:dT) (1 µg/mL) for 6 h. Data are the average luciferase activity ± SD (*p < 0.05; **p < 0.01). (G) Quantitative RT-PCR analysis of HsCCL20 mRNA levels in HEK293T cells transfected with different constructions of DrDDX41 (400 ng/mL) plus pCMV-N-Myc-DrSTING (200 ng/mL) and stimulated with poly(dA:dT) (1 µg/mL) for 6 h. Data are representative of three independent experiments as mean ± SD (*p < 0.05; **p < 0.01). Standard loading was indicated by β-actin expression.