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. Author manuscript; available in PMC: 2016 Mar 15.
Published in final edited form as: J Immunol. 2015 Feb 13;194(6):2826–2837. doi: 10.4049/jimmunol.1402647

Figure 4. Cell-intrinsic IL-25 responses are TRAF4-dependent.

Figure 4

Naïve CD4-positive T-cells were isolated from WT and Traf4 –/– mice and subsequently activated with plate-bound CD3/CD28 and in the indicated polarizing conditions. (a) ELISA for IL-5 and IL-13 performed from supernatants of activated T-cells. (b) RNA was isolated from activated T-cells in Th0+IL-25 conditions and RT-qPCR for the indicated genes normalized to β-actin. (c) Top, Immunoblot from human airway epithelial cell-line (Bet1a) transfected with siRNA against human Traf4 or scrambled (scr) (100 nM each). Bet1a cells transfected with siRNA were treated with hIL-125 (100 ng/ml) for the indicated times, RNA was isolated and RT-qPCR was performed for the indicated genes normalized to GAPDH. (d) Activated T-cells in Th2 polarizing conditions from WT and Traf4 –/– mice were treated with IL-25 (100 ng/ml) for the indicated times, lysates were subjected to SDS-PAGE followed by immunoblotting for the indicated proteins. (e and f) WT and Traf4 –/– or (f) Act1 –/– , KECs were isolated and infected with Ad-V5-Il-17rb (V5-25R). Cells were then stimulated with IL-25 (100 ng/ml) for the indicated timepoints and lysates prepared and subjected to co-immunoprecipitation with antibody against V5. (g and h) HEK293 cells were transfected with the indicated vectors, followed by coimmunoprecipitation for 24 hours using antibodies against tagged IL-25R. Coimmunoprecipitate and whole cell lysate (WCL) were separated by SDS-PAGE and immunoblotted with the indicated antibodies. (i) Il-17rb –/– KECs were transduced with the indicated vectors followed by IL-25 stimulation, RT-qPCR was performed and normalized to β-actin. All data are representative of at least 3 independently performed experiments. Error bars represent mean ± SEM, * indicates p<0.05, ** p<0.01, *** p<0.001.