Journal: Redox Biology

Article Title: AGR2-mediated cell-cell communication controls the antiviral immune response by promoting the thiol oxidation of TRAF3

doi: 10.1016/j.redox.2025.103581

Figure Lengend Snippet: AGR2 interacts with TRAF3 and inhibits TRAF3 K63-linked ubiquitination to disrupt IRF3-dependent signaling A Immunoblot analysis of total and phosphorylated (p-) TBK1, total and phosphorylated (p-) IRF3 in lysates of BEAS-2B cells transfected with pcDNA3.1 or pcDNA3.1-AGR2 infected with VSV for 0, 2, 4h. B Immunoblot analysis of total and phosphorylated (p-) TBK1, total and phosphorylated (p-) IRF3 in lysates of h wild type or Agr2 Tg BMDMs infected with VSV for 0, 2, 4h. C Confocal microscopic images of wild type or Agr2 Tg BMDMs infected with VSV and probed with anti-AGR2(Red), anti-IRF3(Green), and DAPI . D QPCR analysis of IFNB1 mRNA in BEAS-2B cells transfected with the indicated expression plasmids for RIG-IN, MDA5, MAVS, TBK1, or IRF3-5D and AGR2 or control vector, n = 3 samples examined over 3 independent experiments. E Co-IP analysis of the interaction between TRAF3 and TBK1 in HCT-116 cells with (+) or without (−) transfected AGR2 infected with VSV for 2 h. F Co-IP analysis of the interaction between TRAF3 and TBK1 in DLD1 cells with (+) or without (−) knockdown AGR2 infected with VSV for 2 h. G Co-IP analysis of the interaction between AGR2 and TRAF3 in DLD-1 cells. H Co-IP analysis of the interaction between AGR2 and TRAF3 in HEK293T cells co-transfected with Flag-AGR2 and Myc-TRAF3 plasmids. I-J Model of Human TRAF3 domains and truncation mutants ( I ) and binding of Myc-TRAF3 or its truncation mutants with Flag-AGR2 in transiently transfected HEK293T cells, as determined by co-IP and IB analysis ( J ). K Immunoprecipitation (with anti-TRAF3) and immunoblot analysis of lysates of HEK293T cells co transfected for 48 h with plasmid encoding Myc-TRAF3, HA-tagged ubiquitin (HA-Ub), with (+) or without (−) RIG-IN and Falg-AGR2. L-M Immunoprecipitation (with anti-TRAF3) and immunoblot analysis of lysates of HEK293T cells co transfected for 48 h with plasmid encoding Myc-TRAF3, K48-linked ubiquitin (HA-Ub-K48) ( L) , or K63-linked ubiquitin (HA-Ub-K63) ( M ), with (+) or without (−) RIG-IN and Flag-AGR2. Data in (D) are presented as mean ± SD and P -values by two-tailed unpaired Student's t-test are indicated. ns, not significant; ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.

Article Snippet: Antibodies against AGR2 (No. 12275-1-AP), TRAF3 (No. 18099-1-AP), TRAF3 (Clone No. 1E3F4) (No. 66310-1-Ig), TBK1 (No. 28397-1-AP), TBK1 (No. 67211-1-Ig), beta-Actin (No. 81115-1-RR), beta-Tubulin (No. 10068-1-AP), GAPDH (Clone No. 1E6D9) (No. 60004-1-Ig), and mCherry (No. 26765-1-AP) were obtained from Proteintech.

Techniques: Ubiquitin Proteomics, Western Blot, Transfection, Infection, Expressing, Control, Plasmid Preparation, Co-Immunoprecipitation Assay, Knockdown, Binding Assay, Immunoprecipitation, Two Tailed Test

Journal: Redox Biology

Article Title: AGR2-mediated cell-cell communication controls the antiviral immune response by promoting the thiol oxidation of TRAF3

doi: 10.1016/j.redox.2025.103581

Figure Lengend Snippet: AGR2 inhibited TRAF3 K63-linked ubiquitination through thioredoxin motif A Schematic diagram of human AGR2 and its truncation mutants. B Co-IP analysis of the interaction between TRAF3 and AGR2, AGR2 E60A/K64A or AGR2 C81S in HEK293T cells transfected with the plasmids expressing Myc-TRAF3, with Flag-AGR2, Flag-AGR2 E60A/K64A or Flag-AGR2 C81S. C Non-reduced and reduced immunoblots of the lysates of HEK-293T cells transfected with plasmids expressing Flag-TRAF3, with AGR2, AGR2 E60A/K64A or AGR2 C81S. D Co-IP analysis of the interaction between TRAF3 and AGR2 with or without reductant TCEP (2 mM) or b-ME (20 mM) in HEK293T cells transfected with the plasmids expressing Myc-TRAF3, with Flag-AGR2. E Non-reduced and reduced immunoblots of the lysates of HEK-293T cells transfected with plasmids expressing Flag-TRAF3, with AGR2 and treatment with or without reduced. F Immunoprecipitation analysis of ubiquitination of TRAF3 in HEK293T cells transfected with the plasmids expressing Myc-TRAF3, HA-K63 only Ub, and Flag-AGR2, or Flag-AGR2 C81S. G QPCR analysis the expression of IFNB1 mRNA (left panel) or ELISA analysis of IFN-β production (right panel) in vector, AGR2 or AGR2 C81S transfected BEAS-2B cells infected with VSV, n = 3 samples examined over 3 independent experiment. H Immunoblot analysis of total and phosphorylated (p-) TBK1, total and phosphorylated (p-) IRF3 in lysates of Beas-2B cells transfected with Vector, AGR2, or AGR2 C81S infected with VSV for 0, 2, 4h. I QPCR analysis the expression of Ifnb1 mRNA (left panel) or ELISA analysis of IFN-β production (right panel) in BSA, wild type AGR2 protein or AGR2 C81S protein treated BMDMs infected with VSV, n = 3 samples examined over 3 independent experiment. J Immunoblot analysis of total and phosphorylated (p-) TBK1, total and phosphorylated (p-) IRF3 in lysates of BSA, wild type AGR2 protein or AGR2 C81S protein treated BMDMs infected with VSV for 0, 2, 4h. Data in (G and I) are presented as mean ± SD and P -values by one-way ANOVA with Tukey's multiple comparisons test are indicated. ns, not significant; ∗, P < 0.033; ∗∗, P < 0.002; ∗∗∗, P < 0.001.

Article Snippet: Antibodies against AGR2 (No. 12275-1-AP), TRAF3 (No. 18099-1-AP), TRAF3 (Clone No. 1E3F4) (No. 66310-1-Ig), TBK1 (No. 28397-1-AP), TBK1 (No. 67211-1-Ig), beta-Actin (No. 81115-1-RR), beta-Tubulin (No. 10068-1-AP), GAPDH (Clone No. 1E6D9) (No. 60004-1-Ig), and mCherry (No. 26765-1-AP) were obtained from Proteintech.

Techniques: Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Transfection, Expressing, Western Blot, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Infection

Journal: Redox Biology

Article Title: AGR2-mediated cell-cell communication controls the antiviral immune response by promoting the thiol oxidation of TRAF3

doi: 10.1016/j.redox.2025.103581

Figure Lengend Snippet: Cysteine 296 represents a crucial functional regulatory site in TRAF3. A Conservation analysis of TRAF3 Cys296 among species by T-Coffee. B Co-IP analysis of the interaction between AGR2 and TRAF3 or TRAF3 C296S in HEK293T cells transfected with the plasmids expressing AGR2, with Flag-TRAF3 or Flag-TRAF3 C296S. C Non-reduced and reduced immunoblots of the lysates of HEK293T cells transfected with various plasmids, as indicated. D Immunoprecipitation analysis of ubiquitination of TRAF3 in HEK293T cells transfected with the plasmids expressing HA-K63 only Ub, and Myc-TRAF3 or Myc-TRAF3 C296S. E Highest scoring AlphaFold3-Multimer model, modeled using AGR2 residues 1–175 and TRAF3 residues 1–400. F Co-IP analysis of TRAF3 self-association in HEK293T cells transfected with the plasmids expressing Flag-TRAF3, with Flag-TRAF3 or Flag-TRAF3 C296S. G Immunoprecipitation analysis of ubiquitination of TRAF3 in HEK293T cells transfected with the plasmids expressing HA-K63 only Ub, and, Myc-TRAF3, Myc-TRAF3 C68A/H70A, Myc-TRAF3 C296S or Myc-TRAF3 C68A/H70A/C296S. H Co-IP analysis of the interaction between TBK1 and TRAF3 or TRAF3 C296S in HCT-116 cells with (+) or without (−) transfected N-RIG-I. I QPCR analysis the expression of Ifnb1 mRNA in vector, TRAF3 or TRAF3 C296S transfected Traf3 −/− MEFs infected with VSV for 4h, n = 3 samples examined over 3 independent experiment. Data in (I) are presented as mean ± SD and P -values by one-way ANOVA with Tukey's multiple comparisons test are indicated. ∗, P < 0.033; ∗∗∗, P < 0.001.

Article Snippet: Antibodies against AGR2 (No. 12275-1-AP), TRAF3 (No. 18099-1-AP), TRAF3 (Clone No. 1E3F4) (No. 66310-1-Ig), TBK1 (No. 28397-1-AP), TBK1 (No. 67211-1-Ig), beta-Actin (No. 81115-1-RR), beta-Tubulin (No. 10068-1-AP), GAPDH (Clone No. 1E6D9) (No. 60004-1-Ig), and mCherry (No. 26765-1-AP) were obtained from Proteintech.

Techniques: Functional Assay, Co-Immunoprecipitation Assay, Transfection, Expressing, Western Blot, Immunoprecipitation, Ubiquitin Proteomics, Plasmid Preparation, Infection