Fig 2: Degradation of cIAP1 and synthesis of cIAP2 upon H. pylori infection in AGS, ALPK1‐KO, and TIFA‐KO cells AGS, ALPK1‐KO, and TIFA‐KO cells were infected with H. pylori. Total cell lysates were analyzed by immunoblotting for the expression of cIAP1 and cIAP2.AGS cells were treated with 10 µM MG‐132 thirty minutes after infection with H. pylori. Total cell lysates were analyzed by immunoblotting for the expression of cIAP1 and phosphorylation of p100 and IκBα.AGS cells were treated with 10 µM MG‐132 thirty minutes after infection with H. pylori. Cells were harvested in lysis buffer containing 1% SDS. Lysates were diluted 1:10 in lysis buffer without SDS and subjected to IP with antibody against cIAP1 or an isotype‐matched antibody (IgG). Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies. 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.

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.

Fig 4: 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 5: Relative protein and mRNA expression of IAPs in untreated and BV6 treated MCF7 and MDA-MB-231cells determined by Real-time PCR and western blotting, respectively. (a) Western Blot images showing the protein bands of untreated control and treated breast cancer cells with 0.5 μM and 1 μM of BV6. Histogram displaying the fold change in the XIAP cIAP1, cIAP2 and survivin protein expression levels normalized by Actin. (b) Fold change in XIAP, cIAP1, cIAP2, survivin and caspase3 mRNA expression levels normalized by Actin in both breast cancer cell lines. The experiments are carried out in triplicates displayed as mean ± SEM, and the data represented is statistically significant (*), (**), and (***) show p < 0.05, p < 0.01 and p < 0.001, respectively.