Examination of the inhibitory role of TcpAh in TRIF signaling pathway. (A,B) Activation of zebrafish IFNφ1/IFNφ2 promoters detected in zebrafish embryos microinjected with IFNφ1 or IFNφ2 luciferase reporter (IFNφ1 or IFNφ2-Luc; 100 pg/embryo), renilla luciferase reporter (10 pg/embryo), and increasing amounts (0, 50, and 100 pg/embryo) of TcpAh expression vectors under stimulation with PolyI:C (200 pg/embryo) for 12 h. Data are the average luciferase activity ± SD (**p < 0.01; ***p < 0.001). (C,D) Activation of human IRF3 and IFN-β promoters in HEK293T cells transfected with human IRF3 or IFN-β luciferase reporter (IRF3-Luc or IFN-β-Luc; 200 ng/mL), renilla luciferase reporter (15 ng/mL), zebrafish TRIF expression vector (50 ng/mL), and increasing amounts (0, 100, and 500 ng/mL) of TcpAh expression vectors. Data are the average luciferase activity ± SD (**p < 0.01; ***p < 0.001). (E,F) Real-time PCR analysis for the expression of zebrafish IFNφ1 (E) and IFNφ2 (F) in leukocytes, which were sorted from peripheral blood, spleen, and kidney tissues at indicated time after i.p. stimulation with PBS, wild-type A. hydrophila and ΔtcpAh mutant. Data are representative of three independent experiments as mean ± SD (**p < 0.01). Standard loading was indicated by β-actin expression.
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