rabbit anti traf3  (Cell Signaling Technology Inc)

Journal: Biochemistry and Biophysics Reports

Article Title: The miR-2110/ TRAF3 axis is associated with endothelial dysfunction and atherosclerosis in coronary heart disease

doi: 10.1016/j.bbrep.2026.102508

Figure Lengend Snippet: Validation of TRAF3 as a direct target of miR-2110 by RT-qPCR, luciferase reporter assay, and Western blot analysis ( A ) RT-qPCR analysis of TRAF3 mRNA levels in OE2110 and OENC cells, calculated using the 2 −ΔΔCt method, normalized to GAPDH, and expressed relative to the control group (set to 1). ( B ) TRAF3 mRNA levels in peripheral blood samples from healthy controls (n = 30) and CHD patients (n = 34), normalized to GAPDH. ( C ) Schematic representation of the predicted binding sites between miR-2110 and the wild-type (WT) 3′-UTR of TRAF3 , and the corresponding mutated (MT) sequence used for luciferase assays. ( D ) Dual-luciferase reporter assay showing decreased luciferase activity in cells co-transfected with miR-2110 mimics and TRAF3 -WT but not TRAF3 -MT. ( E, F ) Western blot analysis and quantification of TRAF3 protein expression in OE2110 and OENC, with GAPDH as internal reference. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ns: not significant. Panels A, D, and F: Data are presented as mean ± SD from three independent biological experiments (N = 3) using EA.hy926 cells. Panel E: Representative blots are shown. Statistical comparisons were performed using unpaired two-tailed Student's t-tests. Panel B (patient samples): Data were presented as median (interquartile range) and were analyzed using the Mann–Whitney U test.

Article Snippet: Endogenous peroxidase activity was quenched with 3% H 2 O 2 for 10 min at room temperature, followed by blocking with 5% normal goat serum (Solarbio, China, SL038) in PBS for 1 h. Sections were incubated overnight at 4 °C with rabbit anti-TRAF3 monoclonal antibody (1:200, HUABIO, China, PSH01-30), then probed with HRP-conjugated goat anti-rabbit IgG (1:500, Servicebio, China, GB23303) for 1 h at room temperature.

Techniques: Biomarker Discovery, Quantitative RT-PCR, Luciferase, Reporter Assay, Western Blot, Control, Binding Assay, Sequencing, Activity Assay, Transfection, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: Biochemistry and Biophysics Reports

Article Title: The miR-2110/ TRAF3 axis is associated with endothelial dysfunction and atherosclerosis in coronary heart disease

doi: 10.1016/j.bbrep.2026.102508

Figure Lengend Snippet: Increased TRAF3 expression in cardiac and aortic tissues of ApoE −/− mice fed a high-fat diet ( A ) Relative Traf3 mRNA expression in the aortic root and adjacent heart tissue quantified by RT-qPCR, calculated using the 2 −ΔΔCt method, normalized to GAPDH, and expressed relative to the control group (set to 1). ( B, C ) Western blot analysis and quantification of TRAF3 protein expression in heart tissues. ( D, E ) Western blot analysis and quantification of TRAF3 protein expression in aortic tissues. ( F ) Representative immunohistochemical staining of TRAF3 in aortic root sections from control and ApoE −/− HFD mice, showing predominant localization in the tunica intima, including the endothelial layer and subendothelial regions containing vascular smooth muscle cells and infiltrating leukocytes (200 × ). Red boxes highlight regions with enhanced staining in HFD mice. (G) Quantification of TRAF3-positive area in the tunica intima. ∗ P < 0.05. Data represent mean ± SD; n = 6 mice per group. Statistical comparisons were performed using unpaired two-tailed Student's t-tests.

Article Snippet: Endogenous peroxidase activity was quenched with 3% H 2 O 2 for 10 min at room temperature, followed by blocking with 5% normal goat serum (Solarbio, China, SL038) in PBS for 1 h. Sections were incubated overnight at 4 °C with rabbit anti-TRAF3 monoclonal antibody (1:200, HUABIO, China, PSH01-30), then probed with HRP-conjugated goat anti-rabbit IgG (1:500, Servicebio, China, GB23303) for 1 h at room temperature.

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Immunohistochemical staining, Staining, Two Tailed Test

Journal: Autophagy

Article Title: Picornavirus VP2 protein suppresses innate immunity through selective autophagic degradation of IKBKE/IKKε

doi: 10.1080/15548627.2025.2597460

Figure Lengend Snippet: SVA VP2 protein inhibits the expression of IKBKE and interacts with it. (A) HEK-293T cells were transfected with empty vector or Flag-VP2 plasmids. The cells were lysed at 24 hpt and analyzed by western blotting using the indicated antibodies. (B-D) HEK-293T cells were transfected with 0, 0.25, 0.5 or 1 μg of Flag-VP2 expressing plasmids for 24 h. The expression levels of endogenous IKBKE (B), RIGI (C), or IRF3 (D) were assessed by western blotting. (E) HEK-293T cells were transfected with Flag-VP2 expressing plasmids (0, 0.25, 0.5 or 1 μg) for 24 h. Total rna was extracted from the cells, and IKBKE mRNA levels were quantified by qPCR. (F, G) HEK-293T cells were infected with SVA (MOI = 0.5) for 0, 4, 8 and 12 h respectively, and the protein expression of IKBKE was analyzed by western blotting (F), the mRNA expression of IKBKE was detected by qPCR (G). (H) HEK-293T cells were co-transfected with Flag-VP2 and either Vec, various HA-tagged innate immune molecule-expressing plasmids (MDA5, RIGI, MAVS, TRAF6, TBK1, IRF3, IRF7, IKBKE, and tank) or MYC-tagged TRAF3. At 36 hpt, the cell lysates were subjected to co-IP assay analysis. The immunoprecipitated proteins and whole-cell lysates (WCL) were analyzed by western blotting using the specified antibodies. (I) HEK-293T cells were co-transfected with HA-IKBKE and vector or Flag-VP2 expressing plasmids for 36 h. The cell lysates were immunoprecipitated with anti-HA or control IgG antibodies, and the antigen-antibody complex was subjected to western blotting analysis. (J) HEK-293T cells were mock-infected or infected with SVA at an MOI of 0.5 for 12 h. Cell lysates were then immunoprecipitated with anti-VP2 or control IgG antibodies, the antigen-antibody complex were assessed by western blotting. (K) HEK-293T cells were mock-infected or infected with SVA (MOI = 0.1) for 10 h, after which the colocalization of IKBKE (red) and VP2 (green) was assessed by immunofluorescence assay (IFA). Nuclei were counterstained with DAPI (blue). (l, M) HEK-293T cells were co-transfected with increasing amounts of Flag-VP2 and MYC-IKBKE, along with HA-TBK1 (L) or HA-IRF3 (M) expressing plasmids. At 24 hpt, the cells were treated with SeV for another 12 h. The cells were lysed and immunoprecipitated with anti-MYC antibodies. The immunoprecipitated proteins and WCL were analyzed by western blotting using the specified antibodies.

Article Snippet: The commercial antibodies used in this study include: anti-Flag mouse Ab (Sigma, F1804), anti-MYC mouse Ab (Sigma, M5546), anti-HA mouse Ab (Proteintech, 66,006–2-Ig), anti-RIGI rabbit Ab (Cell Signaling Technology, 3743S), anti-IFIH1/MDA5 rabbit Ab (Abcam, Ab126630 ), anti-MAVS rabbit Ab (Cell Signaling Technology, 3993S), anti-TRAF3 rabbit Ab (Cell Signaling Technology, 4729T), anti-TRAF6 rabbit Ab (Cell Signaling Technology, 67591S), anti-TBK1 rabbit Ab (Cell Signaling Technology, 38066S), anti-IRF3 rabbit Ab (Proteintech, 11,312–1-AP), anti-IRF7 rabbit Ab (Abcam, ab109255), anti-LC3 rabbit Ab (Proteintech, 14,600–1-AP), anti-TUBB/β-tubulin mouse Ab (Abclonal, A12289), anti-IKBKE/IKKε rabbit Ab (Abclonal, A3463), anti-K33 rabbit Ab (Abclonal, A18199), anti-RNF114 rabbit Ab (Abclonal, A10636), anti-CALCOCO2/NDP52 rabbit Ab (Abclonal, A24021), anti-SQSTM1/p62 rabbit Ab (Abclonal, A19700).

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Infection, Co-Immunoprecipitation Assay, Immunoprecipitation, Control, Immunofluorescence

Journal: Autophagy

Article Title: Picornavirus VP2 protein suppresses innate immunity through selective autophagic degradation of IKBKE/IKKε

doi: 10.1080/15548627.2025.2597460

Figure Lengend Snippet: SVA VP2 protein inhibits the expression of IKBKE and interacts with it. (A) HEK-293T cells were transfected with empty vector or Flag-VP2 plasmids. The cells were lysed at 24 hpt and analyzed by western blotting using the indicated antibodies. (B-D) HEK-293T cells were transfected with 0, 0.25, 0.5 or 1 μg of Flag-VP2 expressing plasmids for 24 h. The expression levels of endogenous IKBKE (B), RIGI (C), or IRF3 (D) were assessed by western blotting. (E) HEK-293T cells were transfected with Flag-VP2 expressing plasmids (0, 0.25, 0.5 or 1 μg) for 24 h. Total rna was extracted from the cells, and IKBKE mRNA levels were quantified by qPCR. (F, G) HEK-293T cells were infected with SVA (MOI = 0.5) for 0, 4, 8 and 12 h respectively, and the protein expression of IKBKE was analyzed by western blotting (F), the mRNA expression of IKBKE was detected by qPCR (G). (H) HEK-293T cells were co-transfected with Flag-VP2 and either Vec, various HA-tagged innate immune molecule-expressing plasmids (MDA5, RIGI, MAVS, TRAF6, TBK1, IRF3, IRF7, IKBKE, and tank) or MYC-tagged TRAF3. At 36 hpt, the cell lysates were subjected to co-IP assay analysis. The immunoprecipitated proteins and whole-cell lysates (WCL) were analyzed by western blotting using the specified antibodies. (I) HEK-293T cells were co-transfected with HA-IKBKE and vector or Flag-VP2 expressing plasmids for 36 h. The cell lysates were immunoprecipitated with anti-HA or control IgG antibodies, and the antigen-antibody complex was subjected to western blotting analysis. (J) HEK-293T cells were mock-infected or infected with SVA at an MOI of 0.5 for 12 h. Cell lysates were then immunoprecipitated with anti-VP2 or control IgG antibodies, the antigen-antibody complex were assessed by western blotting. (K) HEK-293T cells were mock-infected or infected with SVA (MOI = 0.1) for 10 h, after which the colocalization of IKBKE (red) and VP2 (green) was assessed by immunofluorescence assay (IFA). Nuclei were counterstained with DAPI (blue). (l, M) HEK-293T cells were co-transfected with increasing amounts of Flag-VP2 and MYC-IKBKE, along with HA-TBK1 (L) or HA-IRF3 (M) expressing plasmids. At 24 hpt, the cells were treated with SeV for another 12 h. The cells were lysed and immunoprecipitated with anti-MYC antibodies. The immunoprecipitated proteins and WCL were analyzed by western blotting using the specified antibodies.

Article Snippet: The commercial antibodies used in this study include: anti-Flag mouse Ab (Sigma, F1804), anti-MYC mouse Ab (Sigma, M5546), anti-HA mouse Ab (Proteintech, 66,006–2-Ig), anti-RIGI rabbit Ab (Cell Signaling Technology, 3743S), anti-IFIH1/MDA5 rabbit Ab (Abcam, Ab126630 ), anti-MAVS rabbit Ab (Cell Signaling Technology, 3993S), anti-TRAF3 rabbit Ab (Cell Signaling Technology, 4729T), anti-TRAF6 rabbit Ab (Cell Signaling Technology, 67591S), anti-TBK1 rabbit Ab (Cell Signaling Technology, 38066S), anti-IRF3 rabbit Ab (Proteintech, 11,312–1-AP), anti-IRF7 rabbit Ab (Abcam, ab109255), anti-LC3 rabbit Ab (Proteintech, 14,600–1-AP), anti-TUBB/β-tubulin mouse Ab (Abclonal, A12289), anti-IKBKE/IKKε rabbit Ab (Abclonal, A3463), anti-K33 rabbit Ab (Abclonal, A18199), anti-RNF114 rabbit Ab (Abclonal, A10636), anti-CALCOCO2/NDP52 rabbit Ab (Abclonal, A24021), anti-SQSTM1/p62 rabbit Ab (Abclonal, A19700).

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Infection, Co-Immunoprecipitation Assay, Immunoprecipitation, Control, Immunofluorescence

Journal: Journal of Virology

Article Title: FMDV 3A cooperates with PDCD10 to promote FMDV replication by inhibiting VISA-mediated innate immunity

doi: 10.1128/jvi.00657-25

Figure Lengend Snippet: PDCD10 disrupts VISA-IRF3 complex formation after SeV infection. ( A–E ) Plasmids of PDCD10 (10 µg) and VISA (10 µg) together with RIG-I, TRAF3, TRAF6, TBK1, or IRF3 (10 µg) were transfected into HEK293 cells. After 24 h, cell lysates were subjected to Co-IP. ( F ) PDCD10 was transfected into HEK293 cells with different doses. After 24 h, cell lysates were subjected to Co-IP.

Article Snippet: Primary antibodies against IRF3 (#11904), TRAF6 (#67591), TRAF3 (#33640), TBK1 (#38066), p-IRF3 (#37829), p-TBK1 (#5483), and β-actin (#4970) were purchased from Cell Signaling Technology, Co. Inc. Lipofectamine 2000/3000 was obtained from Invivogene Biotech Co., Ltd., and TRIzol was obtained from Invitrogen (Thermo Fisher Scientific [China] Co., Ltd., China).

Techniques: Infection, Transfection, Co-Immunoprecipitation Assay