Journal: Journal of Biological Chemistry

Article Title: Kaposi's Sarcoma-associated Herpesvirus (KSHV) Oncoprotein K13 Bypasses TRAFs and Directly Interacts with the IκB Kinase Complex to Selectively Activate NF-κB without JNK Activation

doi: 10.1074/jbc.m700118200

Figure Lengend Snippet: FIGURE 3. TRAF2 does not interact with K13 and is not required for the recruitment of K13 to the IKK complex. A and B, cell lysates (C.L.) prepared from Namalwa and BCBL-1cells expressing an empty vector or the indicated FLAG-tagged K13 constructs were immunoprecipitated (I.P.) using control (C) or FLAG (F) antibody beads and co-immunoprecipitated IKK complex subunits were detected by immunoblotting with antibodies against IKK/ and Nemo/IKK, respectively, as described previously (5). The blots were re-probed with TRAF2, TRAF3 and TRAF5 antibodies to check the interaction of K13 with TRAFs. C, cell lysates (C.L.) prepared from BC-3 and BCBL-1 cells expressing an empty vector or FLAG-tagged K13 were immunoprecipitated using control (C) or FLAG (F) beads and the presence of interacting proteins detected by Western blotting with the indicated antibodies. D, 293T cells were transfected with K13-FLAG and cFLIP-FLAG along with either an empty vector or TRAF2-HA. Approximately 36-h post-transfection, cells were lysed, and cell lysates (C.L.) were immunoprecipi- tated using control (C) or FLAG (F) antibody beads and the interaction of cFLIP and K13 with TRAF2 was detected by Western blotting.

Article Snippet: RNA Interference (RNAi)—siRNA oligos against TRAF2 and TRAF3 were purchased from Santa Cruz Biotechnology. p65 and a control siRNAs have been described previously (22). siRNAoligonucleotides (80 nM) were transfected using calcium phosphate, as described previously (23).

Techniques: Expressing, Plasmid Preparation, Construct, Immunoprecipitation, Control, Western Blot, Transfection

Journal: Journal of Biological Chemistry

Article Title: Kaposi's Sarcoma-associated Herpesvirus (KSHV) Oncoprotein K13 Bypasses TRAFs and Directly Interacts with the IκB Kinase Complex to Selectively Activate NF-κB without JNK Activation

doi: 10.1074/jbc.m700118200

Figure Lengend Snippet: FIGURE 5. TRAF2 and TRAF3 are not required for K13-induced NF-B acti- vation. A, a luciferase reporter assay showing equivalent NF-B activation in WT and TRAF2-deficient MEFs by transient transfection of K13. MEFs were transfected with an NF-B/luciferase reporter construct and a Renilla reporter construct (normalization control) and the experiment performed as described previously (14). The data represent mean S.E. of a representative of two independent experiments performed in duplicate with similar results. B and C, EMSA demonstrating equivalent increase in NF-B DNA binding activity (B) and similar NF-B subunit composition (C) in the WT and TRAF2- deficient MEF cells with stable expression of K13. The position of the induced NF-B complexes is marked by an arrowhead, whereas an asterisk marks the position of the constitutive complexes. A supershift assay was performed using a control antiserum (lane 1), or antisera against p50 (lane 2), p65 (lane 3), Rel B (lane 4), and c-Rel (lane 5) subunits of NF-B. D and E, 293-NFB-Luc-K13- ERTAM cells were transfected with a control siRNA (against HTLV-1 Tax) or siRNAs directed against TRAF2, TRAF3 and p65, respectively. Approximately 72-h post-infection, cells were either mock-treated or treated with 4-OHT (20 nM) for 12 h, and cell lysates used for the measurement of luciferase activity (D). The data represent mean S.E. of a representative of two independent experiments with similar results. Immunoblot confirms spe- cific knock-down of TRAF2, TRAF3, and p65 proteins following siRNA-me- diated gene silencing (E).

Article Snippet: RNA Interference (RNAi)—siRNA oligos against TRAF2 and TRAF3 were purchased from Santa Cruz Biotechnology. p65 and a control siRNAs have been described previously (22). siRNAoligonucleotides (80 nM) were transfected using calcium phosphate, as described previously (23).

Techniques: Luciferase, Reporter Assay, Activation Assay, Transfection, Construct, Control, Binding Assay, Activity Assay, Expressing, Infection, Western Blot, Knockdown

Journal: Journal of neuroinflammation

Article Title: HIV-1 Tat C-mediated regulation of tumor necrosis factor receptor-associated factor-3 by microRNA 32 in human microglia.

doi: 10.1186/1742-2094-9-131

Figure Lengend Snippet: Figure 1 Expression of miR-32 increases with Tat C treatment in a dose-dependent manner. (a) CHME3 cells were treated with an increasing dose of HIV-1 Tat C protein. After 24 hours, cells were harvested for RNA isolation and protein lysate preparation. miR-32 assays were performed by quantitative PCR with TaqMan probes and primers specific for human miR-32. Data was normalized to the expression level of the small RNA, RNU24, and results are shown as fold change compared with untreated control. Changes in miR-32 expression level were significant (P ≤0.05). (b), Western blot analysis for tumor necrosis factor receptor-associated factor 3(TRAF3) of the same samples treated with increasing concentrations of Tat C, showing a gradual reduction in TRAF3 protein expression. (c) Western blot image intensity was normalized to β-tubulin. All experiments were performed three times and are presented as mean ± SE. Changes in the level of expression of TRAF3 in response to increasing dose of Tat C were significant (**P ≤0.005, *P ≤0.05) compared with the untreated group.

Article Snippet: The TRAF3 3′ UTR construct in pMirTarget (SC206836; Origene Technologies) and miR-32 construct as pCMV-Mir (SC400329; Origene Technologies) were used.

Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction, Control, Western Blot

Journal: Journal of neuroinflammation

Article Title: HIV-1 Tat C-mediated regulation of tumor necrosis factor receptor-associated factor-3 by microRNA 32 in human microglia.

doi: 10.1186/1742-2094-9-131

Figure Lengend Snippet: Figure 2 HIV-1 Tat C protein downregulates tumor necrosis factor receptor-associated factor 3 (TRAF3) protein expression. (a) Western blot analysis of TRAF3 in CHME3 cells exposed to HIV-1 Tat C protein. Treating CHME3 cells with 500 ng/ml Tat C significantly reduced the cellular TRAF3 protein level. (b) Densitometry analysis of TRAF3, normalized to β-tubulin image density. The change in TRAF3 expression level in the treated group versus the untreated control group was significant (*P ≤0.05). (c) Quantitative PCR analysis of TRAF3 in CHME3 cells exposed to HIV Tat C protein. The graph is representative of three independent experiments. All experiments were performed at least three times and data are presented as mean ± SE. **P ≤0.005.

Article Snippet: The TRAF3 3′ UTR construct in pMirTarget (SC206836; Origene Technologies) and miR-32 construct as pCMV-Mir (SC400329; Origene Technologies) were used.

Techniques: Expressing, Western Blot, Control, Real-time Polymerase Chain Reaction

Journal: Journal of neuroinflammation

Article Title: HIV-1 Tat C-mediated regulation of tumor necrosis factor receptor-associated factor-3 by microRNA 32 in human microglia.

doi: 10.1186/1742-2094-9-131

Figure Lengend Snippet: Figure 4 Overexpresssion of miR-32 suppresses tumor necrosis factor receptor-associated factor 3(TRAF3) protein expression. (a) Western blot analysis for TRAF3 in CHME3 cells after miR-32 overexpression. Plasmid pCMV-miR-32 was transfected into CHME3 cells. The empty vector was used as the negative control. Cell lysates were prepared after 24 hours of transfection, and western blot analysis was performed using anti-TRAF3 antibody. miR-32 overexpression significantly reduced both mRNA and protein levels of TRAF3 (P ≤0.05) (indicated by * in the transfected group) compared with empty vector. (b) Quantitative (q)PCR analysis of miR-32 overexpression in CHME3 cells, using TaqMan miR-32 assay. miR-32 expression was found to be 7.5-fold higher in miR-32-overexpressed cells. (c) Densitometry quantification of TRAF3 normalized to β- tubulin. (d) qPCR analysis for detection of changes in transcript level of TRAF3 after miR-32 overexpression in CHME3 cells. All experiments were performed at least three times and data are presented as mean ± SE.

Article Snippet: The TRAF3 3′ UTR construct in pMirTarget (SC206836; Origene Technologies) and miR-32 construct as pCMV-Mir (SC400329; Origene Technologies) were used.

Techniques: Expressing, Western Blot, Over Expression, Plasmid Preparation, Transfection, Negative Control

Journal: Journal of neuroinflammation

Article Title: HIV-1 Tat C-mediated regulation of tumor necrosis factor receptor-associated factor-3 by microRNA 32 in human microglia.

doi: 10.1186/1742-2094-9-131

Figure Lengend Snippet: Figure 5 Anti-miR-32 transfection rescues tumor necrosis factor receptor-associated factor 3 (TRAF3) protein expression in CHME3 cells. (a) Transfection efficiency of anti-miR, by using Cy3-labeled anti-miR as negative control. (b) Quantitative (q)PCR analysis of cellular miR-32 level after anti-miR-32 transfection, to confirm the suppression of miR-32. The expression level of miR-32 decreased by 40% in cells transfected with anti-miR-32; compared to cells transfected with scrambled anti-miR negative control (*P ≤0.05). (c) Western blot analysis of TRAF3 in CHME3 cells after anti-miR-32 transfection, showing the recovery of TRAF3 expression level in cells treated with anti-miR-32 and anti-miR-32 plus Tat. Anti-miR-32 transfection was performed at a concentration of 100 pmol/l. After 24 hours of anti-miR-32 transfection, a set of transfected cells were treated with 500 ng/ml Tat C protein to augment the cellular expression level of miR-32. (d) Densitometry analysis of TRAF3 normalized to β-tubulin. There was a significant (**P ≤0.005) recovery of TRAF3 expression level.

Article Snippet: The TRAF3 3′ UTR construct in pMirTarget (SC206836; Origene Technologies) and miR-32 construct as pCMV-Mir (SC400329; Origene Technologies) were used.

Techniques: Transfection, Expressing, Labeling, Negative Control, Western Blot, Concentration Assay

Journal: Journal of neuroinflammation

Article Title: HIV-1 Tat C-mediated regulation of tumor necrosis factor receptor-associated factor-3 by microRNA 32 in human microglia.

doi: 10.1186/1742-2094-9-131

Figure Lengend Snippet: Figure 6 miR-32 directly targets the 3′-UTR of tumor necrosis factor receptor-associated factor 3 (TRAF3). (a) Seed sequence in miR-32 and complementary sequence in the 3′ UTR of TRAF3 mRNA showing seven-mer binding in wild-type (WT) TRAF3 3′ UTR. A deletion mutation of 4 base pairs in the 3′ UTR of TRAF3 was generated by site-directed mutagenesis. This alteration in the 3′ UTR sequence of TRAF3 abrogated the interaction of miR-32 and the 3′ UTR of TRAF3, resulting in translational derepression. (b) Luciferase assays were performed by transfecting HeLa cells with pCMV-β-gal (normalization control), WT TRAF3 3′ UTR and mutated (MUT) TRAF3 3′ UTR plasmids, along with pCMV-miR-32 plasmids. Normalized luciferase light units of control cells are presented as 100 units, and relative light units (RLU) of other treatments are shown accordingly. All experiments were performed three times and data are presented as mean ± SE (error bars). ***P ≤0.0005.

Article Snippet: The TRAF3 3′ UTR construct in pMirTarget (SC206836; Origene Technologies) and miR-32 construct as pCMV-Mir (SC400329; Origene Technologies) were used.

Techniques: Sequencing, Binding Assay, Mutagenesis, Generated, Luciferase, Control

Journal: Journal of neuroinflammation

Article Title: HIV-1 Tat C-mediated regulation of tumor necrosis factor receptor-associated factor-3 by microRNA 32 in human microglia.

doi: 10.1186/1742-2094-9-131

Figure Lengend Snippet: Figure 8 Recovery of tumor necrosis factor receptor-associated factor 3 (TRAF3) expression by anti-miR-32 transfection suppresses expression levels of total interferon regulatory factor (IRF)3 and IRF7. (a) CHME3 cells were transfected with Cy3-labeled control anti-miR, anti-miR-32 and anti-miR-32 plus Tat C treatment. Phosphorylated (p)IRF3 level increased after anti-miR-32 treatment, while the total IRF3 level was downregulated in anti-miR-32-transfected cells, showing a positive relationship between cellular TRAF3 level and activation of IRF3. (b) pIRF7 was increased after anti-miR-32 treatment and anti-miR-32 plus Tat C treatment, again showing a positive role of TRAF3 in IRF7 activation. Total IRF7 level was decreased in anti-miR-32-transfected cells showing that recovery of TRAF3 could modulate the transcription of IRF3and IRF7. (c,d) Densitometry analysis of pIRF3, pIRF7, total IRF3 and total IRF7 normalized to β-tubulin. Experiments were performed three times and data are presented as mean ± SE. Results were significant (*P ≤0.05).

Article Snippet: The TRAF3 3′ UTR construct in pMirTarget (SC206836; Origene Technologies) and miR-32 construct as pCMV-Mir (SC400329; Origene Technologies) were used.

Techniques: Expressing, Transfection, Labeling, Control, Activation Assay

Journal: Journal of neuroinflammation

Article Title: HIV-1 Tat C-mediated regulation of tumor necrosis factor receptor-associated factor-3 by microRNA 32 in human microglia.

doi: 10.1186/1742-2094-9-131

Figure Lengend Snippet: Figure 9 Modulation of tumor necrosis factor receptor-associated factor 3 (TRAF3) protein level alters the interferon regulatory factor (IRF)3 and IRF7 mRNA level. (a,b) CHME3 cells were treated with Tat C protein for 24 hours and transfected with miR-32, respectively. Relative fold changes in mRNA levels were determined for IRF3 and IRF7 using quantitative(q)PCR with SYBR green. As a consequence of Tat C treatment and miR-32 overexpression, the transcript expression levels of IRF3 and IRF7 increased. (c) After inhibiting the cellular miR-32 via application of anti-miR, the transcript level of both IRF3 and IRF7 was reduced. In cells treated with anti-miR-32 plus Tat C, the transcript levels of IRF3 and IRF7 were lower than those in control CHME3 cells. All experiments were repeated three times, and data are presented as mean ± SE. Relative change in IRF3 transcript in miR-32 transfected cells compared with empty vector were significant ***P ≤0.0005, **P ≤0.005 and *P ≤0.05 in respective graphs.

Article Snippet: The TRAF3 3′ UTR construct in pMirTarget (SC206836; Origene Technologies) and miR-32 construct as pCMV-Mir (SC400329; Origene Technologies) were used.

Techniques: Transfection, SYBR Green Assay, Over Expression, Expressing, Control, Plasmid Preparation

Journal: Journal of neuroinflammation

Article Title: HIV-1 Tat C-mediated regulation of tumor necrosis factor receptor-associated factor-3 by microRNA 32 in human microglia.

doi: 10.1186/1742-2094-9-131

Figure Lengend Snippet: Figure 10 Proposed model for HIV-1 Tat C-induced, miR-32-mediated post-transcriptional regulation of tumor necrosis factor receptor-associated factor 3 (TRAF3). In response to HIV-1 Tat C exposure of human microglial cells, miR-32 was upregulated, consequently downregulating the protein level of TRAF3 post-transcriptionally by binding to its 3′ untranslated region. The miRNA inhibitor against miR-32, ant-miR-32, reduced the cellular level of miR-32 and rescued the expression level of TRAF3 protein. The cellular expression level of TRAF3 protein had an inverse relationship to the expression level of interferon regulatory factor (IRF)3/7 and this could perturb the expression of inflammatory genes in microglial cells after exposure to HIV-1 Tat C protein.

Article Snippet: The TRAF3 3′ UTR construct in pMirTarget (SC206836; Origene Technologies) and miR-32 construct as pCMV-Mir (SC400329; Origene Technologies) were used.

Techniques: Binding Assay, Expressing

Journal: Journal of Biological Chemistry

Article Title: Nuclear Factor-κB-inducing Kinase (NIK) Contains an Amino-terminal Inhibitor of Apoptosis (IAP)-binding Motif (IBM) That Potentiates NIK Degradation by Cellular IAP1 (c-IAP1)

doi: 10.1074/jbc.m114.587808

Figure Lengend Snippet: FIGURE 2. The NIK IBM is important for reinforcing the interactions within the c-IAPTRAF2TRAF3NIK complex and is necessary for proper E3 ubiquitin ligase targeting of NIK by c-IAP1. A, HEK293T cells were transfected with TRAF3 siRNA to suppress endogenous TRAF3 expression along with plasmids encoding NIK-GFP or NIK (A2G/V3G)-GFP and FLAG-cIAP1 or FLAG-cIAP1 (H588A). Cell lysates were prepared after 48 h of incubation and analyzed by Western blot using anti-GFP, anti-TRAF3, and anti-FLAG-HRP. B, FLAG-cIAP1 constructs with either NIK-GFP or NIK (A2G/V3G)-GFP were transfected in HEK293T cells, and co-IPs were conducted using an anti-NIK antibody. Eluates were immunoblotted by anti-FLAG-HRP for detection of the c-IAP1-NIK interaction. In addition, the levels of endogenous TRAF2 and TRAF3 in the c-IAPTRAFNIK complex were also analyzed by Western blot. The expression levels of NIK, cIAP1, TRAF2, and TRAF3 in input lysates were confirmed by immunoblotting with indicated antibodies. CON, control; EV, empty vector.

Article Snippet: Antibodies used for immunoblotting: NIK (H-248), TRAF2 (C-20), TRAF3 (G-6), GST (Z-5) (Santa Cruz Biotechnologies); TRAF2 (BD Pharmingen); FLAG-HRP, HA-HRP, FLAG M2, and -actin (Sigma); GFP (Novus).

Techniques: Ubiquitin Proteomics, Transfection, Expressing, Incubation, Western Blot, Construct, Control, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: Dual Role of Cancer Epithelial-Specific TRAF3 in Regulating Breast Cancer Cell Survival and Lymphocyte Activity

doi: 10.3390/ijms27104414

Figure Lengend Snippet: TRAF3 is positively correlated with favorable prognosis in breast cancer. ( a ) High TRAF3 mRNA expression levels are associated with better OS (Living vs. Diseased, Mann–Whitney U test), lower disease stage (Bonferroni correction), lower lymph node stage (N) (N0 vs. N1: p = 0.004, Bonferroni correction) and lower tumor stage (T) (T1 vs. T3: p = 0.019, Bonferroni correction) in the TCGA-BRCA cohort. ( b ) High TRAF3 mRNA expression presents with a statistically significant better OS ( p = 0.00405) and DMFS ( p = 0.00729) in the ER-negative breast cancer cohort employed by GOBO, with ER-positive disease presenting a similar association despite not reaching statistical significance.

Article Snippet: The TRAF3 coding sequence was amplified from a pCMV6-TRAF3-GFP (RG210417, Origene, Rockville, MD, USA) vector and cloned into pLenti-EF1a-GFP-2A-Puro (LV067, ABM Inc, Richmond, BC, Canada).

Techniques: Expressing, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Dual Role of Cancer Epithelial-Specific TRAF3 in Regulating Breast Cancer Cell Survival and Lymphocyte Activity

doi: 10.3390/ijms27104414

Figure Lengend Snippet: Forced TRAF3 expression in breast cancer cell lines induces partial EMT and affects cell proliferation. ( a ) Invasion, migration and colony formation assays depicting an opposing phenotype between migratory and proliferative states of MCF7-TRAF3 cells. ( b ) Western blot analyses for the indicated proteins in MDA-MB-231 and MCF-7 cells (control and TRAF3 expressing). ( c ) ICC for the indicated proteins in MCF-7 cells, indicating significant downregulation of key molecules upon TRAF3 expression (ns: no significance; *** p < 0.001 Student’s t -test).

Article Snippet: The TRAF3 coding sequence was amplified from a pCMV6-TRAF3-GFP (RG210417, Origene, Rockville, MD, USA) vector and cloned into pLenti-EF1a-GFP-2A-Puro (LV067, ABM Inc, Richmond, BC, Canada).

Techniques: Expressing, Migration, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Dual Role of Cancer Epithelial-Specific TRAF3 in Regulating Breast Cancer Cell Survival and Lymphocyte Activity

doi: 10.3390/ijms27104414

Figure Lengend Snippet: Identification of interactors, pathways and processes of TRAF3 in breast cancer. ( a ) Volcano plot of significant TRAF3 interactions in MCF-7 cells (FDR < 0.05). ( b ) Top 20 enriched pathways (Metascape) among proteins that interact with TRAF3 in MCF-7 cells with −log10(Padj) > 10 −20 . ( c ) Significantly enriched pathways among genes co-expressed with TRAF3 in the TCGA BRCA cohort. ( d ) Representative BRCA cases from the TCGA cohort presenting with High and Low TILs (upper panel). High TRAF3 mRNA expression is correlated ( p = 0.02, Mann–Whitney U Test) with High stromal TILs in the TCGA BRCA cohort ( n = 200).

Article Snippet: The TRAF3 coding sequence was amplified from a pCMV6-TRAF3-GFP (RG210417, Origene, Rockville, MD, USA) vector and cloned into pLenti-EF1a-GFP-2A-Puro (LV067, ABM Inc, Richmond, BC, Canada).

Techniques: Expressing, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Dual Role of Cancer Epithelial-Specific TRAF3 in Regulating Breast Cancer Cell Survival and Lymphocyte Activity

doi: 10.3390/ijms27104414

Figure Lengend Snippet: TRAF3 expression across cell populations in the scRNA human breast cancer dataset. ( a ) UMAP visualization of 81,389 quality-filtered single cells derived from the Breast Cancer Atlas, colored by cell type annotation. ( b ) Feature plot showing log-normalized TRAF3 expression projected onto the UMAP embedding. ( c ) Violin plots depicting log-normalized TRAF3 expression across each of the cell types. Statistical comparisons were performed using Wilcoxon rank-sum tests, comparing each cell type against all remaining cells, followed by Benjamini–Hochberg correction for multiple testing. Asterisks (*) indicate adj p -values < 0.05. ( d ) Volcano Plot of Differential expression of TRAF3 -positive ( TRAF3 +) vs. negative ( TRAF3 -) Cancer Epithelial (CE) cells. The x-axis represents the log 2 fold change of expression in TRAF3 -positive versus TRAF3 -negative cells, and the y-axis shows the −log 10 adjusted p -value (FDR). Points are colored according to FDR significance, while labels highlight specific immunologically relevant genes, colored according to the following categories: (i) Immunogenicity—Immunogenicity/Antigen Presentation; (ii) MHC-I—MHC class I pathway (CD8 + T-cell recognition); (iii) MHC-II—MHC class II (tumor-intrinsic or antigen-presenting cell mediated); (iv) Checkpoint—Checkpoint blockade/Immune Modulation; (v) Infiltration—Increase immune infiltration into tumors; and (vi) Non-self—Promote tumor cell recognition as “non-self”. Selected genes of interest not in the above categories are colored black (‘Other’ category). ( e ) Gene Ontology (GO) Enrichment Analysis of the filtered top DE genes (FDR < 0.05 & |log2FC| > 0.1) identified via differential expression analysis between TRAF3 + and TRAF3 -cancer epithelial (CE) cells. X-axis represents the Fold Enrichment, and y-axis represents the immune-related Biological Process and Molecular Function GO terms, grouped into clusters based on functional similarity (for the full GO term graph with all the immune and non-immune related GO terms, see ). Dot size is analogous to the number of specific genes associated with each GO term, while their color gradient corresponds to the FDR-adjusted p -value (Q value). Abbreviations used include the following: CE (Cancer Epithelial cells), NE (Normal Epithelial cells), PVL (PeriVascular-Like cells), CAFs (Cancer-Associated Fibroblasts), PR (Positive Regulation), R (Regulation), prd (production), MM (Molecular Mediator), MBP (Macromolecule Biosynthetic Process), MMP (Macromolecule Metabolic Process), CR (Cellular Response), env/tal (environmental), RSP (receptor signaling pathway), SP (signaling pathway), resp. (response), ext. (external), and If-M (interferon-mediated).

Article Snippet: The TRAF3 coding sequence was amplified from a pCMV6-TRAF3-GFP (RG210417, Origene, Rockville, MD, USA) vector and cloned into pLenti-EF1a-GFP-2A-Puro (LV067, ABM Inc, Richmond, BC, Canada).

Techniques: Expressing, Derivative Assay, Quantitative Proteomics, Immunopeptidomics, Functional Assay

Journal: International Journal of Molecular Sciences

Article Title: Dual Role of Cancer Epithelial-Specific TRAF3 in Regulating Breast Cancer Cell Survival and Lymphocyte Activity

doi: 10.3390/ijms27104414

Figure Lengend Snippet: TRAF3 expression in cancer cells affects PBMC subpopulations and cytokine expression. ( a ) FACs analysis of PBMCs co-cultured with MCF7-TRAF3 cells indicates the downregulation of the CD25+CD127low (Tregs) subpopulation of CD4+ T cells. ( b ) FACS analysis of PBMCs co-cultured with MCF7-TRAF3 cells indicates the upregulation of the CD56+CD16- subpopulation of NK-cells. ( c ) Diagrams depicting absolute quantification of IFN-γ, TNF-α and IL-10 in the supernatants of co-cultured PBMCs/MCF7-TRAF3 cells. ( d ) FACs analysis for live/dead MCF-7 breast cancer cells co-cultured with PBMCs depicting a shift from alive to dead cells in the MCF7-TRAF3 cell population in comparison to MCF7-control cells. ( e ) IHC stain for PD-L1 (CD274) on MCF7-control and MCF7-TRAF3. Arrowheads depict PD-L1 expression only on MCF7-control cells. ( f ) Schematic illustration of a proposed model of TRAF3 action in breast cancer epithelial cells and on the surrounding tumor microenvironmental cells.

Article Snippet: The TRAF3 coding sequence was amplified from a pCMV6-TRAF3-GFP (RG210417, Origene, Rockville, MD, USA) vector and cloned into pLenti-EF1a-GFP-2A-Puro (LV067, ABM Inc, Richmond, BC, Canada).

Techniques: Expressing, Cell Culture, Quantitative Proteomics, Comparison, Control, Staining

Journal: Nature Communications

Article Title: E3 ligase Nedd4l promotes antiviral innate immunity by catalyzing K29-linked cysteine ubiquitination of TRAF3

doi: 10.1038/s41467-021-21456-1

Figure Lengend Snippet: a Peritoneal macrophages from wild-type ( Nedd4l +/+ ) and Nedd4l-deficient ( Nedd4l −/− ) mice were infected with VSV (MOI = 10) for indicated time. Phosphorylated and total TBK1, IRF3, p38, JNK, ERK1/2, NF-kB p65, and IκBα were detected by western blot. Intensities of p-TBK1, TBK1, p-IRF3, and IRF3 signals in the three independent experiments were quantified by ImageJ and averages of the signals were shown in graphs. b Wild-type ( Nedd4l +/+ ) and Nedd4l-deficient ( Nedd4l −/− ) peritoneal macrophages were infected with VSV (MOI = 10) for indicated time, followed by immunoprecipitation (IP) with anti-TRAF3 and immunoblot (IB) analysis with antibodies specific for TBK1, cIAP1/2, HECTD3, TRAF3, Nedd4l, and GAPDH (WCL: whole-cell lysates). c IB of Myc-TBK1, Flag-TRAF3, and Myc-Nedd4l co-immunoprecipitated with anti-Flag from lysates of 293T cells transfected with indicated plasmids. d IB of HA-MAVS, Flag-TRAF3, and Myc-Nedd4l co-immunoprecipitated with anti-Flag from lysates of 293T cells transfected with indicated plasmids. Results in ( a – d ) are representative of three independent experiments.

Article Snippet: Recombinant Nedd4l (TP327866) and recombinant TRAF3 (TP318682) were from Origene.

Techniques: Infection, Western Blot, Immunoprecipitation, Transfection

Journal: Nature Communications

Article Title: E3 ligase Nedd4l promotes antiviral innate immunity by catalyzing K29-linked cysteine ubiquitination of TRAF3

doi: 10.1038/s41467-021-21456-1

Figure Lengend Snippet: a IB of HA-MAVS, HA-TBK1, HA-TRAF3, and Myc-Nedd4l co-immunoprecipitated with anti-Myc from lysates of 293T cells transfected with indicated plasmids with HA-tag, Myc-tag, or GAPDH antibodies. b IB of TRAF3 and Nedd4l co-immunoprecipitated with anti-TRAF3 from lysates of C57BL/6 mice peritoneal macrophages infected with VSV (MOI = 10) for indicated time. c In vitro GST-pull-down assay of Nedd4l with GST or GST-fused TRAF3. d IB of TRAF3 and Nedd4l co-immunoprecipitated with Myc-tag-specific antibody from lysates of 293T cells co-transfected with plasmids expressing Flag-TRAF3 and Myc-tagged wild-type Nedd4l or mutant Nedd4l with CW (∆CW),C2 (∆C2), or HECT (∆HECT) domain deleted. Results in ( a – d ) are representative of three independent experiments.

Article Snippet: Recombinant Nedd4l (TP327866) and recombinant TRAF3 (TP318682) were from Origene.

Techniques: Immunoprecipitation, Transfection, Infection, In Vitro, Pull Down Assay, Expressing, Mutagenesis

Journal: Nature Communications

Article Title: E3 ligase Nedd4l promotes antiviral innate immunity by catalyzing K29-linked cysteine ubiquitination of TRAF3

doi: 10.1038/s41467-021-21456-1

Figure Lengend Snippet: a IB of total ubiquitination, K48- and K63-linked ubiquitination of TRAF3 in VSV-infected wild-type ( Nedd4l +/+ ) and Nedd4l-deficient ( Nedd4l −/− ) peritoneal macrophages. b Plasmids expressing Flag-TRAF3, HA-Ub, and Myc-Nedd4l were co-transfected in 293T cells. After 24 h, cells were infected with VSV (MOI = 10) for 12 h. IB analysis of total ubiquitination, K48- and K63-linked ubiquitination of Flag-tagged TRAF3 with indicated antibodies. c IB analysis of TRAF3 incubated with Nedd4l recombinant proteins and ubiquitination reaction components in vitro. d IB analysis of total ubiquitination of Flag-tagged TRAF3 in 293T cells co-transfected with plasmids expressing Flag-TRAF3, HA-Ub, and Myc-tagged wild-type Nedd4l or mutant Nedd4l with indicated antibodies. e IB analysis of ubiquitination of Flag-TRAF3 in 293T cells co-transfected with plasmids expressing Flag-TRAF3, Myc-Nedd4l, and HA-tagged wild-type Ub or HA-tagged mutant Ub (K11R, K29R, K33R, K48R, and K63R) with indicated antibodies. f IB analysis of ubiquitination of Flag-TRAF3 in 293T cells co-transfected with plasmids expressing Flag-TRAF3, Myc-Nedd4l, and HA-tagged wild-type Ub or HA-tagged mutant Ub (K6 only, K11 only, K27 only, K29 only, K33 only, K48 only, K63 only, Ub-K null) with indicated antibodies. Results in ( a – f ) are representative of three independent experiments.

Article Snippet: Recombinant Nedd4l (TP327866) and recombinant TRAF3 (TP318682) were from Origene.

Techniques: Ubiquitin Proteomics, Infection, Expressing, Transfection, Incubation, Recombinant, In Vitro, Mutagenesis

Journal: Nature Communications

Article Title: E3 ligase Nedd4l promotes antiviral innate immunity by catalyzing K29-linked cysteine ubiquitination of TRAF3

doi: 10.1038/s41467-021-21456-1

Figure Lengend Snippet: a IB analysis of ubiquitination of Flag-tagged TRAF3 in 293T cells co-transfected with plasmids expressing Myc-Nedd4l, HA-tagged mutant Ub (K29 only), and Flag-tagged wild-type TRAF3 or mutant TRAF3 (K50R, K273R, K315R, C56R, or C124R). b IB analysis of total ubiquitination, K48- and K63-linked ubiquitination of Flag-tagged TRAF3 in 293T cells co-transfected with plasmids expressing Myc-Nedd4l, HA-tagged wild-type Ub, and Flag-tagged wild-type TRAF3 or mutant TRAF3 (K50R, K273R, K315R, C56R, or C124R). c IB analysis of total ubiquitination, K48- and K63-linked ubiquitination of Flag-tagged TRAF3 in 293T cells co-transfected with plasmids expressing HA-tagged wild-type Ub and Flag-tagged TRAF3, TRAF3 (C56R), TRAF3 (C124R), TRAF3(C56R/C68A/H70A), or TRAF3(C124R/C68A/H70A). d IB analysis of total ubiquitination, K48- and K63-linked ubiquitination of Flag-tagged TRAF3 in 293T cells co-transfected with plasmids expressing HA-tagged wild-type Ub and Flag-tagged wild-type TRAF3 or mutant TRAF3 (C56R, C124R, C53R, C73R, C76R, C117R, H136R, C141R). Results in ( a – d ) are representative of three independent experiments.

Article Snippet: Recombinant Nedd4l (TP327866) and recombinant TRAF3 (TP318682) were from Origene.

Techniques: Ubiquitin Proteomics, Transfection, Expressing, Mutagenesis

Journal: Nature Communications

Article Title: E3 ligase Nedd4l promotes antiviral innate immunity by catalyzing K29-linked cysteine ubiquitination of TRAF3

doi: 10.1038/s41467-021-21456-1

Figure Lengend Snippet: a IB of Flag-cIAP1, Flag-cIAP2, Myc-Nedd4l, and HA-TRAF3 co-immunoprecipitated with anti-HA from lysates of 293T cells transfected with indicated plasmids. b IB of Flag-HECTD3, Myc-Nedd4l and HA-TRAF3 co-immunoprecipitated with anti-HA from lysates of 293T cells transfected with indicated plasmids. c IB of Flag-cIAP1, Flag-TRAF3, and Flag-TRAF3(C56R) co-immunoprecipitated with anti-TRAF3 from lysates of 293T cells transfected with indicated plasmids. d IB of Flag-HECTD3, Flag-TRAF3 and Flag-TRAF3(C56R) co-immunoprecipitated with anti-TRAF3 from lysates of 293T cells transfected with indicated plasmids. e RT-qPCR analysis of IFN-β, IL-6, and TNF-α mRNA expression in 293 cells transfected with plasmids expressing TRAF3 or mutant TRAF3 and then infected with VSV (MOI = 10) for 12 h. Data are presented as mean ± SD ( n = 3 per group) and p -values by two-tailed unpaired Student’s t -test are indicated in e. Results in ( a – e ) are representative of three independent experiments.

Article Snippet: Recombinant Nedd4l (TP327866) and recombinant TRAF3 (TP318682) were from Origene.

Techniques: Immunoprecipitation, Transfection, Quantitative RT-PCR, Expressing, Mutagenesis, Infection, Two Tailed Test