Fig 1: NEMO mediates recruitment of TBK1 and IKKe to inhibit IL-17RSC complex assembly and downstream signaling A, BST2 wild-type or NEMO KO cells were stimulated for 15 min with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. (A) The principal component analysis of two independent experiments. (B) The ratio between iBAQ intensities of selected IL-17RSC components to iBAQ intensity of IL-17RC. Mean + SEM from two independent experiments is shown.C, DNEMO-deficient ST2 cells were reconstituted with NEMO(WT), NEMO (?201–248) lacking TANK/NAP1-binding site or empty vector and stimulated with SF-IL-17 (500 ng/ml) for 15 min or were left unstimulated and IL-17 was added post-lysis. Lysates were subjected to anti-Flag immunoprecipitation to isolate IL-17RSC (C) or tested for the activation of signaling pathways (D) and samples were analyzed by immunoblotting.ENEMO-deficient ST2 cells were reconstituted with NEMO(WT) or empty vector, treated or not with TBK1/IKKe inhibitor MRT67307 (2 µM) and stimulated with SF-IL-17 (500 ng/ml) as indicated. Activation of signaling pathways was analyzed upon cell lysis by immunoblotting.F, GHeLa wild-type or NEMO KO cells were stimulated with SF-IL-17 (500 ng/ml) for 15 min or were left unstimulated and IL-17 was added post-lysis. Lysates were subjected to anti-Flag immunoprecipitation to isolate IL-17RSC (F) or tested for the activation of signaling pathways (G) and samples were analyzed by immunoblotting.HNEMO-deficient HeLa cells were reconstituted with NEMO(WT) or empty vector and stimulated with IL-17 (500 ng/ml) as indicated. Cells were solubilized and analyzed by immunoblotting.Data information: Immunoblot results are representative of two (C, D, E, G, H) or four (F) independent experiments. Source data are available online for this figure.
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