Fig 1: Regulation of FlaA-induced antibacterial effectors by Stat.(A) Analysis of the promoters of MjCtl556 and MjAlf2238. The 5′ untranslated regions were obtained and analyzed using the online Promoter 3.0 tool and JAPAR tool. (B) Stat phosphorylation induced by V. anguillarum or rFlaA, with PBS or rTag as control, respectively. Stat phosphorylation was detected using a human Phospho-STAT5A-Y694 antibody. Left panel, representative blotting result; right panel, quantification and statistical analysis of the blotting data from three repeats. (C) Influence of MjShoc2 knockdown on Stat-activation caused by FlaA. rFlaA injection was performed 24 h after dsRNA application, and Stat phosphorylation was detected another 6 h later. Left panel, representative blotting result; right panel, quantification and statistical analysis of the blotting data from three repeats. (D) RNAi efficiency of Stat. Expression was detected by Western blotting at 24 h after dsRNA application. Upper panel, representative blotting result; lower panel, quantification and statistical analysis of the blotting data from three repeats. (E) Influence of Stat knockdown on the induction of MjCtl556 and MjAlf2238 expression by FlaA. rFlaA injection was performed 24 h after dsRNA application, and the expression levels were detected 6 h later. Data are mean ± SD from three repeats. (F) ChIP assay showing the binding of Stat to MjCtl556 and MjAlf2238 promoters. Shrimp were injected with rFlaA or the tag. The hemocytes were collected as the pool for the ChIP assay at 6 h later. The immunoprecipitates were analyzed using RT-PCR with primers specific for the fragments containing the STAT5 binding sites. Data are representative of three repeats. (G) Influence of MjShoc2 knockdown on FlaA-induced binding of Stat to MjCtl556 and MjAlf2238 promoters. The number shows relative ratio of the band intensity (anti-Stat/input) of each panel. Data are representative of three repeats. Each sample originated from at least five animals. *, 0.01 < p < 0.05, **, 0.001< p < 0.01, ***, p < 0.001.