Fig 2: Co‐localization of proteins involved in the activation of classical and alternative NF‐κB pathways with the TIFAsomes in H. pylori infection A–DIn TIFA‐KO cells transiently expressing TIFA‐tdTomato, co‐localization of TRAF6 (A), TRAF2 (B), cIAP1 (C), and TRAF3 (D) with TIFA‐tdTomato, which formed TIFAsomes upon infection with H. pylori P1 wild‐type strain, were detected by immunofluorescence staining. The nuclei were counterstained with DAPI. Scale bar = 10 µm.ETIFA‐KO cells transiently expressing TIFA‐tdTomato were infected with H. pylori P1 strain mutated in the gmhA gene (ΔHP0857). Scale bar = 10 µm. Data information: Data shown are representative for at least two independent experiments.

Fig 3: Induction of the classical and alternative NF‐κB pathways by H. pylori depends on ADP‐heptose A, BInfection of NCI‐N87 and HeLa cells with the H. pylori P1 wild‐type or isogenic ΔHP0857 (mutated in the gmhA gene) strains was performed.CAGS cells were infected with the H. pylori P1 wild‐type strain, isogenic strain mutated in the gmhA gene (ΔHP0857) alone or complemented with P1 gmhA gene. Asterisk denotes an unspecific band and the arrow indicates the band corresponding to phosphorylated p100.DInfection of AGS, TIFA‐KO, and ALPK1‐KO cells with the H. pylori P12 wild‐type strain was performed. Asterisk denotes an unspecific band and the arrow indicates the band corresponding to phosphorylated p100. Data information: Total cell lysates were analyzed by immunoblotting with the indicated antibodies. Data shown are representative for at least two independent experiments. GAPDH served as loading control. Source data are available online for this figure.

Fig 4: Co‐localization of TRAF6 with TIFAsomes formed upon H. pylori infection TIFA‐KO cells transiently expressing TIFA‐tdTomato were left uninfected or infected with H. pylori for 60 min. TIFAsomes were detected by epifluorescence microscopy. Secondary antibody control with no primary antibody added was also shown. The nuclei were counterstained with DAPI. Scale bar = 10 µm.TIFA‐KO cells were transiently transfected with TIFA‐tdTomato and infected with H. pylori for 60 min, followed by immunofluorescence detection of TRAF6. Specific regions of interest (ROIs) in the micrographs were analyzed for co‐localization between TRAF6 and 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 (left) and 10 µm (right).

Fig 5: ALPK1 and TIFA are required for induction of the classical NF‐κB pathway in H. pylori infection AParental AGS, ALPK1‐KO, and TIFA‐KO cells were infected with H. pylori for the times shown. Total cell lysates were analyzed by immunoblotting using the indicated antibodies. CagA demonstrated a similar infection rate of the different cells. Asterisk denotes an unspecific band and the arrow indicates the band corresponding to phosphorylated p100.BTotal RNA was isolated from ALPK1‐KO cells and analyzed using quantitative RT‐PCR for the ALPK1 transcript. Data shown depict the average of triplicate determinations normalized to the RPL13A housekeeping gene. Error bars denote RQ ± RQmin/RQmax.CTIFA‐KO cells were transfected with His‐tagged recombinant TIFA protein 24 h prior to H. pylori infection. Co‐IP with an anti‐His antibody was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies. Asterisk denotes an unspecific band and the arrow indicates the TRAF2 band.D, EAGS and ALPK1‐KO (D) or TIFA‐KO (E) cells were infected with H. pylori for the times shown. Co‐IP with an anti‐TRAF6 antibody or isotype‐matched antibody (IgG) was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies.FAGS and TIFA‐KO cells were treated with 30 ng/ml IL‐1β for the times shown. Co‐IP with an anti‐TRAF6 antibody or isotype‐matched antibody (IgG) was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies. Data information: Data shown (A, C‐F) are representative for at least two independent experiments. GAPDH served as loading control. Data shown in (B) are from one experiment with three technical repeats. Source data are available online for this figure.