anti-helicobacter pylori cag antigen igg fraction  (AMS Biotechnology)

Journal: EMBO Reports

Article Title: TIFA has dual functions in Helicobacter pylori ‐induced classical and alternative NF‐κB pathways

doi: 10.15252/embr.202152878

Figure Lengend Snippet: A AGS cells were infected with H. pylori or treated with 20 ng/ml LTα 1 β 2 for the indicated times. Total cell lysates were analyzed by immunoblotting using the indicated antibodies. B, C AGS cells were infected with H. pylori P1 wild‐type or isogenic ΔHP0857 (mutated in the gmhA gene) strains. Total cell lysates were analyzed by immunoblotting using the indicated antibodies. D AGS cells were treated with 200 nM synthetic ADP‐heptose for the indicated times. Total cell lysates were analyzed by immunoblotting using the indicated antibodies. E AGS cells were pre‐incubated with 100 μM dynamin GTPase inhibitor Dynasore for 60 min followed by H. pylori infection or addition of ADP‐heptose (200 nM). Total cell lysates were analyzed by immunoblotting using the indicated antibodies. CagA demonstrated a similar infection rate. 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.

Article Snippet: The following primary antibodies were used: GAPDH (Millipore, #MAB374); CagA (Austral Biologicals, #HPM‐5001‐5); Flagellin (Acris, AM00865PU‐N); C23 (sc‐13057), A20 (sc‐166692), RelA (sc‐8008), RelB (sc‐226), cIAP1 (sc‐271419, for denaturing IP), TRAF2 (sc‐136999), and His‐probe (sc‐804) were purchased from Santa Cruz Biotechnology; phospho‐IκBα (#9246), IκBα (#4812), phospho‐RelA (#3031), phospho‐p38 (#4511), p38 (#9212), phospho‐SAPK/JNK (#4671), phospho‐p44/42 (Erk1/2) (#4377), phospho‐p100 (#4810), phospho‐TAK1 (#4508), NIK (#4994), TIFA (#61358), TAK1 (#5206), TRAF6 (#8028), SAPK/JNK (#9252), cIAP1 (#7065), cIAP2 (#3130), p44/42 MAPK (Erk1/2) (#4696), and p100/p52 (#4882) were purchased from Cell Signaling Technology.

Techniques: Infection, Western Blot, Incubation

Journal: EMBO Reports

Article Title: TIFA has dual functions in Helicobacter pylori ‐induced classical and alternative NF‐κB pathways

doi: 10.15252/embr.202152878

Figure Lengend Snippet: A Parental 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. B Total 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 ± RQ min /RQ max . C TIFA‐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, E AGS 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. F AGS 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.

Article Snippet: The following primary antibodies were used: GAPDH (Millipore, #MAB374); CagA (Austral Biologicals, #HPM‐5001‐5); Flagellin (Acris, AM00865PU‐N); C23 (sc‐13057), A20 (sc‐166692), RelA (sc‐8008), RelB (sc‐226), cIAP1 (sc‐271419, for denaturing IP), TRAF2 (sc‐136999), and His‐probe (sc‐804) were purchased from Santa Cruz Biotechnology; phospho‐IκBα (#9246), IκBα (#4812), phospho‐RelA (#3031), phospho‐p38 (#4511), p38 (#9212), phospho‐SAPK/JNK (#4671), phospho‐p44/42 (Erk1/2) (#4377), phospho‐p100 (#4810), phospho‐TAK1 (#4508), NIK (#4994), TIFA (#61358), TAK1 (#5206), TRAF6 (#8028), SAPK/JNK (#9252), cIAP1 (#7065), cIAP2 (#3130), p44/42 MAPK (Erk1/2) (#4696), and p100/p52 (#4882) were purchased from Cell Signaling Technology.

Techniques: Infection, Western Blot, Isolation, Quantitative RT-PCR, Transfection, Recombinant, Co-Immunoprecipitation Assay

Journal: EMBO Reports

Article Title: TIFA has dual functions in Helicobacter pylori ‐induced classical and alternative NF‐κB pathways

doi: 10.15252/embr.202152878

Figure Lengend Snippet: A The nuclear translocation of transcription factors RelA and RelB was analyzed in nuclear fractions of AGS and TIFA‐KO cells. C23 served as loading control. B Analysis of the activation of ERK1/2, p38, and JNK in AGS and TIFA‐KO cells by immunoblot. CagA demonstrated a similar infection rate of different cells. C–E Analysis of IL‐1β (C), TNF (D), and LTα 1 β 2 (E) treatment of AGS and TIFA‐KO cells by immunoblotting revealed the specificity of TIFA for H. pylori infection. Cell lysates were analyzed by immunoblotting with the indicated antibodies. Asterisk denotes an unspecific band and the arrow indicates the band corresponding to phosphorylated p100 (E). F Transfection of His‐tagged recombinant TIFA into TIFA‐KO cells rescued the activation of classical and alternative NF‐κB pathways upon H. pylori infection. Cell lysates were analyzed by immunoblotting with the indicated antibodies. G AGS cells were transfected with siRNA against TRAF2 and infected with the H. pylori P1 wild‐type strain. Cell lysates were analyzed by immunoblotting using the indicated antibodies. H AGS cells were transfected with siRNA against TRAF6 and infected with the H. pylori P1 wild‐type strain. 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.

Article Snippet: The following primary antibodies were used: GAPDH (Millipore, #MAB374); CagA (Austral Biologicals, #HPM‐5001‐5); Flagellin (Acris, AM00865PU‐N); C23 (sc‐13057), A20 (sc‐166692), RelA (sc‐8008), RelB (sc‐226), cIAP1 (sc‐271419, for denaturing IP), TRAF2 (sc‐136999), and His‐probe (sc‐804) were purchased from Santa Cruz Biotechnology; phospho‐IκBα (#9246), IκBα (#4812), phospho‐RelA (#3031), phospho‐p38 (#4511), p38 (#9212), phospho‐SAPK/JNK (#4671), phospho‐p44/42 (Erk1/2) (#4377), phospho‐p100 (#4810), phospho‐TAK1 (#4508), NIK (#4994), TIFA (#61358), TAK1 (#5206), TRAF6 (#8028), SAPK/JNK (#9252), cIAP1 (#7065), cIAP2 (#3130), p44/42 MAPK (Erk1/2) (#4696), and p100/p52 (#4882) were purchased from Cell Signaling Technology.

Techniques: Translocation Assay, Activation Assay, Western Blot, Infection, Transfection, Recombinant