Examination of the inhibitory role of TcpAh in MyD88 signaling pathway. (A–C) Activation of the NF-κB-binding promoters detected in zebrafish embryos microinjected with NF-κB luciferase reporter (NF-κB-Luc; 100 pg/embryo), renilla luciferase reporter (10 pg/embryo), and increasing amounts (0, 50, and 100 pg/embryo) of TcpAh expression vectors with stimulation of (A) Pam3CSK4 (200 pg/embryo), (B) CpG-ODN (400 pg/embryo), and (C) TNFα (10 pg/embryo) for 12 h. Data are the average luciferase activity ± SD (*p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant). (D) Activation of the NF-κB-binding promoter detected in HEK293T cells transfected with NF-κB luciferase reporter (NF-κB-Luc; 150 ng/mL), renilla luciferase reporter (15 ng/mL), MyD88 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 IL-1β (E) and TNFα (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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