anti traf4 antibody  (Cell Signaling Technology Inc)

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: RNA binding protein Sam68 promotes germinal center reaction and IgG response through regulation of miR29

doi: 10.1007/s00018-026-06145-w

Figure Lengend Snippet: Sam68 regulates Traf4 expression in B cells through mir29. A - B . Real time quantitative PCR (RT-qPCR) analysis measuring expression of miR29a, b and c (A) and Traf4 (B) in WT (black bars) and Sam68 KO (red bars) splenic B cells at different time points after CD40 stimulation. Bars represent mean ± SEM ( n = 3–4 independent experiments). Statistical analysis: Two-way ANOVA, multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001. C . Flow cytometric analysis showing the electroporation efficiency of empty vector (EV) and pR miR29a in WT B cells as detected by DsRed/mRFP expression. % of DsRed or mRFP. + cells are indicated in the figure. D - E . RT-qPCR analysis measuring expression of miR29a (D) and Traf4 (E) in WT B cells electroporated either with EV or pR miR29a (29a OE). Bars represent mean ± SEM ( n = 3). Statistical analysis: one sample t-test. * p < 0.05. F . Representative western blot showing Traf4 expression in WT B cells overexpressing miR29a (29a OE) compared to EV expressing cells. Traf4 over-expressing WT B cells (Traf4 OE) are used as positive control. Lower band- endogenous Traf4, upper band- 3X FLAG tagged overexpressed Traf4 (see methods and Supplementary Fig. for details). Gapdh is used as loading control. G. Quantification of the band intensities of the western blot shown in F. Bars represent mean ± SEM ( n = 3). Statistical analysis: One way ANOVA, multiple comparison test. * p < 0.05. H. Flow cytometric analysis showing the retroviral transduction efficiency of EV and mTraf4 in WT and Sam68 KO B cells as detected by DsRed expression. % of DsRed + cells are indicated in the figure. I. Proliferation analysis of WT and Sam68 KO B cells overexpressing either EV (upper panel) or Traf4 protein (bottom panel) 48 h post stimulation with IL4 + anti-CD40 antibody. Green histograms indicate the different generation of cells as modeled by proliferation modeling in Flowjo. J. Quantification of proliferation parameters for WT and KO cells overexpressing EV (open bars) or Traf4 (T4, dotted bars). Bars represent mean ± SEM ( n = 3–4). Statistical analysis: One way ANOVA, multiple comparison test. * p < 0.05, ** p < 0.01

Article Snippet: For Traf4 gene expression analysis, 50 ng of Trizol RNA was reverse transcribed using high capacity RNA to cDNA kit (#4387406, Thermo Fisher) and gene expression was measured by Taqman assay using the probe Traf4 (Mm00493829_m1) and Taqman Gene expression master mix (#4369016, Thermo Fisher) following manufacturer’s protocol.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Comparison, Electroporation, Plasmid Preparation, Western Blot, Positive Control, Control, Retroviral, Transduction

Journal: Cellular & Molecular Biology Letters

Article Title: PDIA6 promotes the cell proliferation of ESCC by enhancing the disulfide bond formation in TRAF4

doi: 10.1186/s11658-026-00876-1

Figure Lengend Snippet: PDIA6 binds with TRAF4 in ESCC cells. A Pull down–mass spectrometry assay identified candidate PDIA6-interacting proteins in KYSE450 PDIA6-OE cells compared with the control group. B Exogenous co-immunoprecipitation (Co-IP) assays in 293T cells confirmed the interaction between PDIA6 and TRAF4. C In vitro co-immunoprecipitation (Co-IP) assays using purified proteins confirmed the interaction between PDIA6 and TRAF4. D BiFC assay demonstrated the direct binding of PDIA6 to TRAF4 in 293T cells. Scale bars: 50 μm. E , F Endogenous Co-IP in KYSE30 ( E ) and KYSE70 ( F ) cells revealed PDIA6 binding to TRAF4. G , H Endogenous Co-IP in KYSE30 ( G ) and KYSE70 ( H ) cells revealed that TRAF4 could bind to PDIA6. I Protein–protein docking predicted the interaction interface between PDIA6 and the N-terminal domain of TRAF4. PDIA6 is shown in pink and TRAF4 in blue. J Schematic of the full-length TRAF4 and truncated domains with the RING finger domain (1–277 aa) or TRAF domain (278–470). Co-IP assays showed PDIA6 bound to the TRAF4’s 1–277 domain

Article Snippet: The purified PCR fragments were then ligated into the enzyme-digested vectors, including pLVX-IRES-Puro-3 × Flag, pcDNA3.1/V5-His A, pCMV-myc, and pcDNA3.1/V5-His A for TRAF4 constructs using T4 DNA ligase (M0202L, New England Biolabs, USA).

Techniques: Mass Spectrometry, Control, Immunoprecipitation, Co-Immunoprecipitation Assay, In Vitro, Purification, Bimolecular Fluorescence Complementation Assay, Binding Assay

Journal: Cellular & Molecular Biology Letters

Article Title: PDIA6 promotes the cell proliferation of ESCC by enhancing the disulfide bond formation in TRAF4

doi: 10.1186/s11658-026-00876-1

Figure Lengend Snippet: PDIA6 enhances the formation of disulfide bonds of TRAF4. A Chemical modification assay using DTT, NEM, and mPEG5000-Mal revealed the presence of disulfide bonds in full-length TRAF4. B The truncated V5-TRAF4 protein (1–277 aa) was treated with DTT, NEM, and mPEG5000-Mal, resulting in a molecular weight shift, indicating that disulfide bonds were formed in the 1–277 domain. C The truncated V5-TRAF4 protein (278–470 aa) was subjected to the same chemical modification treatment, but no significant molecular weight change was observed, suggesting the absence of disulfide bonds in this region. D 293T cells were transfected with different truncated V5-tagged TRAF4 plasmids and treated with a cysteine-specific modifier to detect disulfide bond-forming regions of TRAF4 by Western blot. E The TRAF4 AlphaFold model indicated the locations of cysteines with high scores. F Chemical modification of TRAF4 mutants identified Cys39/42 and Cys83/106 as key residues for disulfide bond formation. G Ubiquitination assay demonstrated that C42A and C83A mutations reduce TRAF4-mediated AKT ubiquitination. H Co-IP assays showed that DTT treatment disrupted the interaction between PDIA6 and TRAF4, indicating disulfide-bond-dependent binding. I Co-IP assays using TRAF4 mutants (C39/42 or C83/106) demonstrated that mutation of these cysteine residues similarly abolished the PDIA6–TRAF4 interaction

Article Snippet: The purified PCR fragments were then ligated into the enzyme-digested vectors, including pLVX-IRES-Puro-3 × Flag, pcDNA3.1/V5-His A, pCMV-myc, and pcDNA3.1/V5-His A for TRAF4 constructs using T4 DNA ligase (M0202L, New England Biolabs, USA).

Techniques: Modification, Molecular Weight, Transfection, Western Blot, Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Binding Assay, Mutagenesis

Journal: Cellular & Molecular Biology Letters

Article Title: PDIA6 promotes the cell proliferation of ESCC by enhancing the disulfide bond formation in TRAF4

doi: 10.1186/s11658-026-00876-1

Figure Lengend Snippet: PDIA6 increases the stability of TRAF4 by inhibiting the ubiquitination of TRAF4 in ESCC cells. A The protein levels of TRAF4 were measured by Western blot in PDIA6 knockout KYSE30 and KYSE70 cells. B The mRNA levels of TRAF4 were measured by qPCR in KYSE30 and KYSE70 cells with PDIA6 knockout. C , D PDIA6-knockout KYSE30 ( C ) and KYSE70 ( D ) cells were treated with 50 μg/mL CHX for 0, 6, or 8 h, and TRAF4 degradation was analyzed by western blot. E , F The TRAF4 degradation in KYSE30 ( E ) and KYSE70 ( F ) cells after treatment with CHX, MG132 (20 μM), or CQ (20 μM). G The ubiquitination assays showed that SMURF1 promoted TRAF4 ubiquitination. H Ubiquitination assays showed that PDIA6 suppressed SMURF1-mediated ubiquitination of TRAF4. I Dose-dependent overexpression of PDIA6 gradually reduced the interaction between TRAF4 and SMURF1. J The ubiquitination assays showed that TRAF4 mutants C39/42 and C83/106 exhibited increased ubiquitination. K The ubiquitination assays showed that the PDIA6-mediated inhibition of TRAF4 ubiquitination was impaired in C39/42 and C83/106 mutants, highlighting the importance of disulfide bonds in stabilizing TRAF4

Article Snippet: The purified PCR fragments were then ligated into the enzyme-digested vectors, including pLVX-IRES-Puro-3 × Flag, pcDNA3.1/V5-His A, pCMV-myc, and pcDNA3.1/V5-His A for TRAF4 constructs using T4 DNA ligase (M0202L, New England Biolabs, USA).

Techniques: Ubiquitin Proteomics, Western Blot, Knock-Out, Over Expression, Inhibition

Journal: Cellular & Molecular Biology Letters

Article Title: PDIA6 promotes the cell proliferation of ESCC by enhancing the disulfide bond formation in TRAF4

doi: 10.1186/s11658-026-00876-1

Figure Lengend Snippet: PDIA6 promotes ESCC cell proliferation through the TRAF4/AKT1/mTOR signaling pathway. A The levels of TRAF4 protein in various human ESCC cell lines. B The efficiency of TRAF4 knockout in KYSE30 and KYSE70 cells was assessed using Western blot. C Cell viability was measured using cell proliferation assays on stable TRAF4 knockout KYSE30 and KYSE70 cells at the designated time points. D The images and statistical analysis of soft agar assays from KYSE30 and KYSE70 cells after TRAF4 knockout. E The images and statistical analysis of the colony formation assays conducted on TRAF4 knockout cells in KYSE30 and KYSE70. F , G The protein levels of AKT signaling pathway components, including p-AKT, p-p70S6K, and p-mTOR, were detected by Western blot after knockout of TRAF4 ( F ) and PDIA6 ( G ) ESCC cells. H The protein levels of AKT signaling pathway components were evaluated using western blot after restoring TRAF4 WT and C42A/C83A in ESCC cells with PDIA6 knockout. I Cell proliferation assays were used to measure the proliferation ability of PDIA6 knockout cells after restoring TRAF4 WT, C42A, and C83A proteins J The images of colony formation assay after TRAF4 WT and C42A/C83A were restored in PDIA6 knockout cells. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The purified PCR fragments were then ligated into the enzyme-digested vectors, including pLVX-IRES-Puro-3 × Flag, pcDNA3.1/V5-His A, pCMV-myc, and pcDNA3.1/V5-His A for TRAF4 constructs using T4 DNA ligase (M0202L, New England Biolabs, USA).

Techniques: Knock-Out, Western Blot, Colony Assay