Fig 2: Co-localization of TRAF3, TRAF2, or cIAP1 with TIFAsomes formed upon H. pylori infectionTIFA-KO cells were transiently transfected with TIFA-tdTomato and infected with H. pylori for 60 min, followed by immunofluorescence detection of TRAF2, cIAP1, and TRAF3. Specific regions of interest (ROIs) in the micrographs were analyzed for co-localization of TRAF2, cIAP1, or TRAF3 with TIFAsomes. The Fiji software with the plug-in JACoP using Costes’ automatic threshold was used. Presented are the values for Pearson’s coefficient (r) and Manders’s coefficient (M1&M2). Scale bar = 20 µm (TRAF2, cIAP1) and 10 µm (TRAF3).

Fig 3: ALPK1-mediated TIFA oligomerization is required for induction of the alternative NF-?B pathway in H. pylori infection TIFA-KO cells were transfected with His-tagged recombinant TIFA protein 24 h prior to H. pylori infection or treatment with 30 ng/ml LTa1ß2. Co-IP with an anti-His antibody was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies.AGS cells were pre-incubated with 100 µM dynamin GTPase inhibitor Dynasore for 60 min followed by addition of ADP-heptose (200 nM). Co-IP with an anti-TIFA antibody was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies. Asterisk indicates an unspecific band and the arrow indicates the TRAF3 band.AGS and ALPK1-KO cells were infected with H. pylori for the times shown. Co-IP with an anti-TIFA antibody or isotype-matched antibody (IgG) was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies.AGS and TIFA-KO cells were infected with H. pylori for the times shown. Co-IP with an anti-TRAF2 antibody or isotype-matched antibody (IgG) was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies.AGS cells were transfected with siRNA against cIAP1. Co-IP with an anti-TIFA antibody or isotype-matched antibody (IgG) was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies.TIFA-KO cells were transfected with empty vector plasmid (pCMV6), wild-type human (phTIFA), phosphorylation-defective mutant (pT9A), or TRAF6 binding-defective mutant (pE178A) FLAG-tagged TIFA plasmids. Total cell lysates were analyzed by immunoblotting using the indicated antibodies.TIFA-KO cells were transfected with empty vector plasmid (pCMV6), wild-type human (phTIFA), phosphorylation-defective mutant (pT9A), or TRAF6 binding-defective mutant (pE178A) FLAG-tagged TIFA plasmids. Total cell lysates were separated by native gel electrophoresis under non-reducing conditions. Data information: Data shown are representative for at least two independent experiments. GAPDH served as loading control. Source data are available online for this figure.