Fig 1: Alternative NF-κB signaling contributes to cell survival in H. pylori infection. a AGS cells were transfected with non-target-specific siRNA (scr) or siRNA targeting RelB (RelBsi_E1) for 48 h. After H. pylori infection for 24 h, cells were harvested and stained with annexin V-FITC/PI, and analyzed by flow cytometry. A graph showing data from three independent experiments is depicted (means ± SD). ##p < 0.005, *p < 0.01 and **p < 0.001. Representative dot plots are shown for each treatment. b A proposed model for A20’s role in the negative regulation of alternative NF-κB signaling pathway in H. pylori infection. H. pylori-induced activation of the classical NF-κB pathway results in the up-regulation of A20 expression. A20 binds to TIFA in the TIFA/NIK regulatory complex, which in turn destabilizes the association of TIFA with the complex. This restores the stability of cIAP1 in the complex and this TIFA-free NIK regulatory complex recovers the ability to mediate the proteasomal degradation of NIK, leading to the shutdown of alternative NF-κB signaling
Fig 2: Inhibition of H. pylori-mediated alternative NF-?B signaling pathway by A20 requires TIFA. a WT and A20KO_1 AGS cells were infected with H. pylori. Total cell lysates were harvested and IP was performed using an antibody against TRAF2 or isotype-matched IgG (IgG). The numbers indicate the band intensities of cIAP1 (normalized to the respective band intensities of TRAF2) of H. pylori-infected samples relative to uninfected control. b WT and TIFA-knockout (TIFAKO) AGS cells were infected with H. pylori or treated with 30 ng/ml LTa1ß2. Total cell lysates were subjected to IP as in (a). c AGS cells were transfected with non-target-specific siRNA (scr) or siRNAs targeting TRAF2 (TRAF2si) or cIAP1 (cIAP1si_8) for 48 h prior to infection with H. pylori. Total cell lysates were used for IP with an antibody against TIFA or isotype-matched IgG. d Following incubation of recombinant human A20 and TIFA proteins in vitro, IP was performed using an antibody against TIFA. e as in (d) but IP was performed using an antibody against A20 or isotype-matched IgG. f A20KO_1 AGS cells were infected with H. pylori and IP was performed as in (c). IB analyses were performed with TIFA-immunoprecipitates without and with one hour incubation with 100 ng recombinant A20 protein prior to elution of immunoprecipitates. A representative blot of at least two experiments was shown
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