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: After blocking with 5% skimmilk in PBS, the membranes were immunoblotted overnight at 4 °C with antibodies against TRAF3 (1:2000, Proteintech, USA, 18099-1-AP) and GAPDH (1:50,000, Proteintech, USA, 10494-1-AP), followed by HRP-conjugated goat Anti-rabbit IgG (H + L) (1:10000, Servicebio, China, GB23303).

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: After blocking with 5% skimmilk in PBS, the membranes were immunoblotted overnight at 4 °C with antibodies against TRAF3 (1:2000, Proteintech, USA, 18099-1-AP) and GAPDH (1:50,000, Proteintech, USA, 10494-1-AP), followed by HRP-conjugated goat Anti-rabbit IgG (H + L) (1:10000, Servicebio, China, GB23303).

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

Journal: Microbiology Spectrum

Article Title: The segmented flavivirus ALSV-encoded nucleoprotein VP2 inhibits type I interferon production by targeting RIG-I

doi: 10.1128/spectrum.02484-25

Figure Lengend Snippet: ALSV VP2 suppresses IFN-I production by targeting the upstream of TBK1. ( A–D ) HEK293T cells were transfected with an IFN-β-luc reporter plasmid, a control plasmid, and plasmids expressing ALSV VP2 protein, along with MAVS ( A ), TRAF3 ( B ), TBK1 ( C ), and IRF3 ( D ) expressing plasmids to induce IFN-I production. At 24 hpt, cells were subjected to the luciferase activity assay and immunoblotting analysis using the phosphorylation and total antibodies of IRF3 and TBK1, along with the tag antibodies. Gray-scale statistical analysis of phosphorylation relative to total protein is conducted. ( E and F ) HEK293T cells were transfected with plasmids expressing ALSV VP2 protein, along with the indicated signaling molecules. At 48 hpt, cells were subjected to immunoblotting analysis. The relative levels of the indicated proteins normalized to β-Actin are shown. Data from independent experiments ( n ≥ 3) were statistically analyzed using two-way ANOVA with multiple comparison correction (*** P < 0.001 and **** P < 0.0001).

Article Snippet: The following primary antibodies were utilized: glyceraldehyde 3-phosphate dehydrogenase (GAPDH; ProteinTech, cat#10494-1-AP), HA (ProteinTech, cat#51064-2-AP), GST (ProteinTech, cat#10000-0-AP), Flag (ProteinTech, cat#20543-1-AP), Myc (ProteinTech, cat#60003-2-Ig), β-Actin (ProteinTech, cat#66009-1-Ig), IFIT3 (ProteinTech, cat#15201-1-AP), IFIT1 (Cell Signaling Technology, cat#14769), Phospho-IRF3 (Abways, cat#CY6575), Phospho-TBK1 (Cell Signaling Technology, cat#5483s), IRF3 (ProteinTech, cat#11312-1-AP), TBK1 (Abcam, cat#AB40676), RIG-I (ProteinTech, cat#20566-1-AP), MDA5 (ProteinTech, cat#21775-1-AP), MAVS (ProteinTech, cat#14341-1-AP), TRAF3 (ProteinTech, cat#18099-1-AP), LC3 (ProteinTech, cat#14600-1-AP), and ATG5 (HUABIO, cat#ET1611-38).

Techniques: Transfection, Plasmid Preparation, Control, Expressing, Luciferase, Activity Assay, Western Blot, Phospho-proteomics, Comparison

Journal: Microbiology Spectrum

Article Title: The segmented flavivirus ALSV-encoded nucleoprotein VP2 inhibits type I interferon production by targeting RIG-I

doi: 10.1128/spectrum.02484-25

Figure Lengend Snippet: ALSV VP2 interacts with RIG-I to suppress its activity. ( A ) HEK293T cells were transfected with Flag-VP2 and HA-tagged RIG-I, TBK1, TRAF3, or an empty vector. At 48 hpt, cells were subjected to anti-HA immunoprecipitates and analyzed by immunoblotting. ( B ) HEK293T cells were transfected with Flag-VP2 and Myc-tagged MAVS or an empty vector. At 48 hpt, cells were subjected to anti-Myc immunoprecipitates and analyzed by immunoblotting. ( C ) HEK293T cells were transfected with Flag-VP2 and HA-tagged RIG-I, TBK1, TRAF3, MDA5, or an empty vector. At 48 hpt, anti-HA immunoprecipitates were analyzed by immunoblotting. ( D ) HEK293T cells were transfected with Flag-VP2 and Myc-tagged MAVS or an empty vector. At 48 hpt, cells were subjected to anti-Flag immunoprecipitates and analyzed by immunoblotting. ( E ) HEK293T cells were transfected with Flag-VP2, along with HA-RIG-I, HA-TBK1, HA-TRAF3, or Myc-IRF3. At 48 hpt, cells were subjected to anti-Flag immunoprecipitates and analyzed by immunoblotting. ( F ) HEK293T cells were transfected with Flag-VP2 or an empty vector. At 48 hpt, anti-Flag immunoprecipitates were analyzed by immunoblotting with the indicated endogenous antibodies.

Article Snippet: The following primary antibodies were utilized: glyceraldehyde 3-phosphate dehydrogenase (GAPDH; ProteinTech, cat#10494-1-AP), HA (ProteinTech, cat#51064-2-AP), GST (ProteinTech, cat#10000-0-AP), Flag (ProteinTech, cat#20543-1-AP), Myc (ProteinTech, cat#60003-2-Ig), β-Actin (ProteinTech, cat#66009-1-Ig), IFIT3 (ProteinTech, cat#15201-1-AP), IFIT1 (Cell Signaling Technology, cat#14769), Phospho-IRF3 (Abways, cat#CY6575), Phospho-TBK1 (Cell Signaling Technology, cat#5483s), IRF3 (ProteinTech, cat#11312-1-AP), TBK1 (Abcam, cat#AB40676), RIG-I (ProteinTech, cat#20566-1-AP), MDA5 (ProteinTech, cat#21775-1-AP), MAVS (ProteinTech, cat#14341-1-AP), TRAF3 (ProteinTech, cat#18099-1-AP), LC3 (ProteinTech, cat#14600-1-AP), and ATG5 (HUABIO, cat#ET1611-38).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Western Blot

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: Cell Insight

Article Title: HTATSF1 regulates innate antiviral immune response by orchestrating TRAF3-IRF3 and TRAF6-NF-κB pathways

doi: 10.1016/j.cellin.2025.100294

Figure Lengend Snippet: HTATSF1 interacts with TRAF3 and TRAF6. (A) HTATSF1 is associated with TRAF3 and TRAF6. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids before co-IP and immunoblotting analysis with the indicated antibodies. (B) Endogenous HTATSF1 is constitutively associated with TRAF3 and TRAF6. THP-1 cells (2 × 10 7 ) were left uninfected or infected with HSV-1 (MOI = 5) for 3 or 6 h before co-IP and immunoblotting analysis with the indicated antibodies.

Article Snippet: Antibodies against HA (TA180128) (OriGene); Flag (F3165) and β-actin (A2228) (Sigma); p-TBK1 S172 (ab109272), TBK1 (ab40676), p-IRF3 S386 (ab76493), TAK1 (ab109526), TRAF6 (ab33915), p65 (ab7970), ubiquitin (ab7254), K48-linkage specific polyubiquitin (ab140601) and K63-linkage specific polyubiquitin (ab179434) (Abcam); IRF3 (sc-33641) and STAT1 (SC-417) (Santa Cruz Biotechnology); Myc (5605), p-IRF3 S396 (4947), p-STAT1 Y701 (9167), TRAF3 (4729), p-TAK1 T184/187 (4508), p-IκBα S32/36 (9246), IκBα (9242), p-IKKα/β S176/177 (2078), IKKβ (2370), and p-p65 S536 (3033) (Cell Signaling Technology), HTATSF1 (20805-1-AP) and HECTD3 (11487-1-AP) (Proteintech) were purchased from the indicated companies.

Techniques: Transfection, Co-Immunoprecipitation Assay, Western Blot, Infection

Journal: Cell Insight

Article Title: HTATSF1 regulates innate antiviral immune response by orchestrating TRAF3-IRF3 and TRAF6-NF-κB pathways

doi: 10.1016/j.cellin.2025.100294

Figure Lengend Snippet: Domain mapping of HTATSF1 and TRAF3/6 interactions. (A) & (B) Domain mapping experiments. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids for 18 h before co-IP and immunoblotting analysis with the indicated antibodies. (C) HTATSF1 UHM is essential for its role in promoting innate antiviral signaling. HTATSF1-deficient THP-1 cells were reconstituted with full length HTATSF1 or HTATSF1 UHM. Control THP-1 cells (NC) cells (1 × 10 6 ) were reconstituted with an empty vector. The cells were then stimulated with HSV-1 (MOI = 1) or SeV (MOI = 1) for 6 h before RT-qPCR measurements of mRNA levels of IFNB1 and ISG56 genes. Data shown are mean ​± ​SEM ( n ​= ​3 technical replicates) from one representative experiment, which were repeated for at least two times with similar results. ∗∗, P < 0.01 (unpaired t -test).

Article Snippet: Antibodies against HA (TA180128) (OriGene); Flag (F3165) and β-actin (A2228) (Sigma); p-TBK1 S172 (ab109272), TBK1 (ab40676), p-IRF3 S386 (ab76493), TAK1 (ab109526), TRAF6 (ab33915), p65 (ab7970), ubiquitin (ab7254), K48-linkage specific polyubiquitin (ab140601) and K63-linkage specific polyubiquitin (ab179434) (Abcam); IRF3 (sc-33641) and STAT1 (SC-417) (Santa Cruz Biotechnology); Myc (5605), p-IRF3 S396 (4947), p-STAT1 Y701 (9167), TRAF3 (4729), p-TAK1 T184/187 (4508), p-IκBα S32/36 (9246), IκBα (9242), p-IKKα/β S176/177 (2078), IKKβ (2370), and p-p65 S536 (3033) (Cell Signaling Technology), HTATSF1 (20805-1-AP) and HECTD3 (11487-1-AP) (Proteintech) were purchased from the indicated companies.

Techniques: Transfection, Co-Immunoprecipitation Assay, Western Blot, Control, Plasmid Preparation, Quantitative RT-PCR

Journal: Cell Insight

Article Title: HTATSF1 regulates innate antiviral immune response by orchestrating TRAF3-IRF3 and TRAF6-NF-κB pathways

doi: 10.1016/j.cellin.2025.100294

Figure Lengend Snippet: HTATSF1 promotes K63-linked polyubiquitination of TRAF3 by HECTD3. (A) HTATSF1 promotes K63-linked polyubiquitination of TRAF3. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids for 18 h before ubiquitination assays with the indicated antibodies. WT, wild type; KR, K is mutated to R; KO, K only. (B) HTATSF1 does not regulate polyubiquitination of TBK1. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids for 18 h before ubiquitination assays with the indicated antibodies. (C) HTATSF1-deficiency decreases K63- but not K48-linked polyubiquitination of TRAF3. The control (g NC ) and HTATSF1-deficient (g HTATSF1 ) THP-1 cells (2 × 10 7 ) were left uninfected or infected with HSV-1 (MOI = 5) or SeV (MOI = 5) for the indicated time before ubiquitination assays with the indicated antibodies. (D) HTATSF1 promotes K63-linked polyubiquitination of TRAF3 at K138. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids for 18 h before ubiquitination assays with the indicated antibodies. (E) HECTD3 interacts with TRAF3 but not TRAF2 or TRAF6. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids for 18 h before co-IP and immunoblotting analysis with the indicated antibodies. (F) HTATSF1-deficiency decreases the interactions between TRAF3 and HECTD3 or TBK1. The control (g NC ) and HTATSF1-deficient (g HTATSF1 ) THP-1 cells (2 × 10 7 ) were left uninfected or infected with HSV-1 (MOI = 5) for 6 h before co-IP and immunoblotting analysis with the indicated antibodies. (G) HTATSF1-deficiency attenuates HECTD3-mediated K63-linked polyubiquitination of TRAF3. The control (g NC ) and HTATSF1-deficient (g HTATSF1 ) THP-1 cells (2 × 10 7 ) were transfected with the indicated plasmids for 18 h after PMA (100 ng/mL)-induced adhesion. The cells were then left uninfected or infected with HSV-1 (MOI = 5) for 6 h before co-IP and immunoblotting analysis with the indicated antibodies. (H) HECTD3-deficiency decreases K63-linked polyubiquitination of TRAF3. The control (g NC ) and HECTD3-deficient (g HECTD3 ) THP-1 cells (2 × 10 7 ) were left uninfected or infected with HSV-1 (MOI = 5) or SeV (MOI = 5) for the indicated time before ubiquitination assays with the indicated antibodies.

Article Snippet: Antibodies against HA (TA180128) (OriGene); Flag (F3165) and β-actin (A2228) (Sigma); p-TBK1 S172 (ab109272), TBK1 (ab40676), p-IRF3 S386 (ab76493), TAK1 (ab109526), TRAF6 (ab33915), p65 (ab7970), ubiquitin (ab7254), K48-linkage specific polyubiquitin (ab140601) and K63-linkage specific polyubiquitin (ab179434) (Abcam); IRF3 (sc-33641) and STAT1 (SC-417) (Santa Cruz Biotechnology); Myc (5605), p-IRF3 S396 (4947), p-STAT1 Y701 (9167), TRAF3 (4729), p-TAK1 T184/187 (4508), p-IκBα S32/36 (9246), IκBα (9242), p-IKKα/β S176/177 (2078), IKKβ (2370), and p-p65 S536 (3033) (Cell Signaling Technology), HTATSF1 (20805-1-AP) and HECTD3 (11487-1-AP) (Proteintech) were purchased from the indicated companies.

Techniques: Transfection, Ubiquitin Proteomics, Control, Infection, Co-Immunoprecipitation Assay, Western Blot