Fig. 1.

The SEFIR3 region is critical for the interaction between the Act1 SEFIR domain and IL-17RA. (A) Schematic of wild-type (WT) mAct1 and mAct1 SEFIR deletion mutants. (B) HeLa cells were transiently cotransfected with plasmids encoding FLAG-tagged mouse Act1 or its deletion mutants together with plasmid encoding V5-tagged IL-17RA. Lysates of transfected cells were subjected to immunoprecipation (IP) with antibody against the V5 tag, after which they were analyzed by Western blotting (IB) with antibodies against FLAG and V5. WCL, whole-cell lysate. (C) Act1^−/− MEFs expressing IL-17RB were reconstituted with either empty vector, FLAG-tagged mAct1, or FLAG-tagged deletion mutants of mAct1 by retroviral infection, after which they were treated with IL-17 (50 ng/ml) or IL-25 (100 ng/ml) for 3 hours. The abundances of Cxcl1 and Il13 messenger RNAs (mRNAs) were measured by real-time RT-PCR and the results are expressed as fold-induction calculated as a ratio of the abundance of a given mRNA in the treated sample to that in the untreated sample. The experiment was repeated five times and the data are shown as the mean ± the standard error of the mean (SEM). All other experiments were performed three times, with representative blots shown.