Journal: OncoTargets and therapy

Article Title: RIG-I Promotes Cell Death in Hepatocellular Carcinoma by Inducing M1 Polarization of Perineal Macrophages Through the RIG-I/MAVS/NF-κB Pathway

doi: 10.2147/OTT.S258450

Figure Lengend Snippet: Overexpression of RIG-I activates NF-κB pathway in macrophages. ( A ) Peritoneal macrophages were infected with RIG-I lentivirus or negative control lentivirus and subsequently treated with 100 ng/mL LPS and 20 ng/mL IFN-γ for 24 hours. The levels of RIG-I, MAVS, TRAF2, TRAF3, p65 and IκBα were detected by Western blotting, as well as the phosphorylated p65 and IκBα. ( B ) The gray value of each band was analyzed with Image J software. The expression of RIG-I, MAVS, TRAF2, TRAF3, p65 and IκBα were shown in histogram. **p<0.01, compared with control group.

Article Snippet: Anti-TRAF2 Homo sapiens TNF receptor-associated factor 2 (TRAF2) (cat. no. TA319201) is a rabbit polyclonal antibody and was purchased from OriGene (Beijing, China), and the dilution was 1 μg/mL.

Techniques: Over Expression, Infection, Negative Control, Western Blot, Software, Expressing, Control

Journal: Journal of Advanced Research

Article Title: Metrnl ameliorates diabetic cardiomyopathy via inactivation of cGAS/STING signaling dependent on LKB1/AMPK/ULK1-mediated autophagy

doi: 10.1016/j.jare.2022.10.014

Figure Lengend Snippet: Metrnl promotes the interaction of STING with TRAF2 in mitochondria to induce STING ubiquitination and degradation in cardiomyocytes. ( A ) Effects of Metrnl on TRAF2 in mitochondria. ( B ) Effects of Metrnl shRNA on TRAF2 in mitochondria. ( C ) Confocal images showing the colocalization of STING with TRAF2 in cardiomyocytes. ( D ) Co-IP showing the interaction of STING with TRAF2. ( E ) Effects of Metrnl on the STING/TRAF2 complex. ( F ) Effects of Metrnl shRNA on the STING/TRAF2 complex. ( G ) A proteasome inhibitor MG132 prevented the suppressive effects of Metrnl on STING protein expression. ( H ) Half-life of STING in different groups. ( I ) Effects of Metrnl (left) or Metrnl shRNA (right) on the ubiquitination of STING. ( J ) OE of LKB1 prevented the actions of Metrnl shRNA on the ubiquitination of STING. ( K ) STING siRNA and TRAF2 siRNA weakened the effects of Metrnl on the p62 and LC3 proteins. ( L ) STING siRNA and TRAF2 siRNA weakened the effects of Metrnl on the formation of autophagosome as assessed by LC3 dots. n = 4. * P < 0.05 versus 0 h, NG, Con, or Con shRNA, † P < 0.05 versus HG + Con shRNA, Metrnl or HG, ‡ P < 0.05 versus HG + Metrnl + Con siRNA.

Article Snippet: A scrambled siRNA (100 nM, sc-37007, Santa Cruz, Dallas, TX, USA), ULK1 siRNA (100 nM, SR514693, OriGene Technologies, Inc, Rockville, MD, USA), LKB1 siRNA(100 nM, sc-270074, Santa Cruz, Dallas, TX, USA), STING siRNA (100 nM, SR507303, OriGene Technologies), TRAF2 siRNA (100 nM, SR505994, OriGene Technologies) were transfected to cardiomyocytes using Lipofectamine 2000.

Techniques: Ubiquitin Proteomics, shRNA, Co-Immunoprecipitation Assay, Expressing

Journal: Blood

Article Title: Constitutive BR3 receptor signaling in diffuse, large B-cell lymphomas stabilizes nuclear factor-?B-inducing kinase while activating both canonical and alternative nuclear factor-?B pathways

doi: 10.1182/blood-2010-06-290437

Figure Lengend Snippet: NIK kinase protein accumulates in DLBCL tumor cells. (A) Cell lysates of normal/activated B cells and DLBCL cell lines were probed with NIK, TRAF2, TRAF3, BR3, c-IAP1, or c-IAP2 antibodies in Western blot analyses. Actin was used as a loading control. Box to the right indicates lighter exposure. (B) Cell lysates of DLBCL lymphoma patient biopsies were probed with NIK and BR3 antibodies in Western blot analysis. Actin was used as a loading control. Box to the right indicates lighter exposure. (C) DLBCL TMA analyzed for NIK and BR3 protein expression. Immunohistochemical analysis of DLBCL patient sample TMA of 43 primary DLBCL biopsies using antibodies against NIK and BR3 proteins. Tissue arrays with duplicate samples were scored using an automated analysis system for percentage of positive cells (> 30%) staining for NIK and BR3 protein in the DLBCL subgroups GCB, ABC, and non-profiled (NP; supplemental Figure 1). (D) Representative DLBCL TMA stained for NIK and BR3. Images are representative of the biopsy cores from the DLBCL TMA slides.

Article Snippet: Unless otherwise specified, monoclonal and polyclonal antibodies against the following molecules were used: BLyS, p52, p50, p65, and phospho-p65 (Millipore); NIK and phospho-IκBα (Cell Signaling Technology); polyclonal TRAF3, c-IAP1, and c-IAP2 (Santa Cruz Biotechnology); TRAF3 (BD Pharmingen); TRAF2 (Imgenex); BR3 (Alexis Biochemicals); BR3 antibody 9.1 (Genentech); polyclonal BR3 (ProSci); and human BLyS antibody for neutralization (R&D Systems).

Techniques: Western Blot, Control, Expressing, Immunohistochemical staining, Staining

Journal: Blood

Article Title: Constitutive BR3 receptor signaling in diffuse, large B-cell lymphomas stabilizes nuclear factor-?B-inducing kinase while activating both canonical and alternative nuclear factor-?B pathways

doi: 10.1182/blood-2010-06-290437

Figure Lengend Snippet: Constitutive BLyS/BR3 signaling activates NIK-induced NF-κB pathway activation through induction of TRAF3 degradation in DLBCL. (A) Cytoplasmic cell lysates from MS-GCB or HB-ABC DLBCL cells were immunoprecipitated with BR3 or TRAF3 antibodies and probed with TRAF3 or BR3 antibodies. IgG was used as a negative control. (B) DLBCL-HB cells were co-stained with TRAF3 (FITC, green) or BR3 (Texas Red, red) antibody and analyzed by confocal microscopy. Yellow color indicates co-localization of BR3 and the other probed proteins. (C) Total cell lysates from DLBCL cells (MS) transfected with specific BR3 shRNA or control shRNA were probed with TRAF3 or BR3 antibodies by Western blot analysis. Actin was used as a loading control. Relative protein level of each target molecule was measured with ImageJ densitometer software and normalized to the actin level. (D) Total cell lysates from normal Go B cells stimulated with or without human recombinant BLyS ligand were probed with TRAF3 antibody by Western blot analysis. Actin was used as a loading control. Relative protein level of each target molecule was measured with ImageJ densitometer software and normalized to the actin level. (E) Cytoplasmic extract from DLBCL cells (MS) was subjected to immunoprecipitation analysis with BR3 antibody and then probed with TRAF2, TRAF3, c-IAP1, c-IAP2, or BR3 antibodies by Western blot analysis. (F) DLBCL cells (HB) were co-stained with BR3 (Texas Red, red), along with TRAF2 (FITC, green), c-IAP1 (FITC, green), or c-IAP2 (FITC, green) antibodies, and then analyzed by confocal microscopy. Yellow color indicates co-localization of BR3 and the other probed proteins. (G) Cytoplasmic extracts purified from DLBCL cells (MS) treated with BLyS antibody or control cells were probed with TRAF3 BR3 or NIK antibody in Western blot analysis (left). These samples were also subjected to immunoprecipitation analysis with BR3 antibodies and then probed with TRAF3 or BR3 antibodies for Western blot analysis (right). (H) Cell lysates purified from DLBCL-MS cells transfected with c-IAP1, c-IAP2 shRNA, or control shRNA were analyzed by Western blot analysis with c-IAP1, c-IAP2, or TRAF3 antibody. Actin was used as loading control. Relative protein level of each target molecule was measured with ImageJ densitometer software and normalized to the actin level.

Article Snippet: Unless otherwise specified, monoclonal and polyclonal antibodies against the following molecules were used: BLyS, p52, p50, p65, and phospho-p65 (Millipore); NIK and phospho-IκBα (Cell Signaling Technology); polyclonal TRAF3, c-IAP1, and c-IAP2 (Santa Cruz Biotechnology); TRAF3 (BD Pharmingen); TRAF2 (Imgenex); BR3 (Alexis Biochemicals); BR3 antibody 9.1 (Genentech); polyclonal BR3 (ProSci); and human BLyS antibody for neutralization (R&D Systems).

Techniques: Activation Assay, Immunoprecipitation, Negative Control, Staining, Confocal Microscopy, Transfection, shRNA, Control, Western Blot, Software, Recombinant, Purification

Journal: Frontiers in Endocrinology

Article Title: A novel link between chronic inflammation and humanin regulation in children

doi: 10.3389/fendo.2023.1142310

Figure Lengend Snippet: Local effects of TNF on humanin, PCNA, SOX9 and TRAF2 expressions in growth plates tissue specimens (n=3) in patient 1 and patient 2 analyzed separately. Quantitative analysis of (A, E) humanin staining, (B, F) PCNA staining, (C, G) SOX9 staining, and (D, H) TRAF2 staining. Error bars indicate mean ± SE. Students t-test was used to analyze differences between groups.

Article Snippet: Next, slides were incubated with anti-humanin antibody (NB100-56877; Novus Biologicals, Littleton, Colorado, USA), anti-proliferating cell nuclear antigen (PCNA) antibody (ab-18197; Abcam, Cambridge, United Kingdom), anti-SOX9 antibody (ab-5355; Sigma-Aldrich, Burlington, MA, USA), and anti-TRAF2 antibody (NB100-56173SS; Novus Biologicals, Littleton, Colorado, USA) overnight at 4°C, 1:300 diluted for all antibodies.

Techniques: Staining

Journal: Frontiers in Endocrinology

Article Title: A novel link between chronic inflammation and humanin regulation in children

doi: 10.3389/fendo.2023.1142310

Figure Lengend Snippet: TNF suppressed SOX9, PCNA and TRAF2 expressions in human growth plate tissue specimens (n=6) obtained from 2 children or human chondrocytes. (A, B) Quantitative analysis of SOX9 staining (yellow arrows), calculated as number of positive cells per mm². (C) Relative expression of SOX9 assessed by qPCR in HCS-2/8 cells treated for 72 hours with TNF at 10, 30, 100, 300 ng/ml concentrations (n=3). (D, E) Quantitative analysis of PCNA staining (yellow arrows), calculated as number of positive cells per mm². (F) Relative expression of PCNA assessed by qPCR in HCS-2/8 cells treated for 72 hours with TNF at 10, 30, 100, 300 ng/ml concentrations (n=3). (G) Western blot analysis of SOX9 and PCNA expressions in HCS-2/8 cells treated with TNF (100 ng/ml). (H, I) Quantification of SOX9 and PCNA expressions by three independent Western blot experiments. (J, K) Quantitative analysis of TRAF2 staining (yellow arrows), calculated as number of positive cells per mm². Error bars indicate mean ± SE. Students t-test was used to analyze differences between groups.

Article Snippet: Next, slides were incubated with anti-humanin antibody (NB100-56877; Novus Biologicals, Littleton, Colorado, USA), anti-proliferating cell nuclear antigen (PCNA) antibody (ab-18197; Abcam, Cambridge, United Kingdom), anti-SOX9 antibody (ab-5355; Sigma-Aldrich, Burlington, MA, USA), and anti-TRAF2 antibody (NB100-56173SS; Novus Biologicals, Littleton, Colorado, USA) overnight at 4°C, 1:300 diluted for all antibodies.

Techniques: Staining, Expressing, Western Blot

Journal: Cellular and Molecular Life Sciences

Article Title: A20 undermines alternative NF-κB activity and expression of anti-apoptotic genes in Helicobacter pylori infection

doi: 10.1007/s00018-022-04139-y

Figure Lengend Snippet: Inhibition of H. pylori -mediated alternative NF-κB signaling pathway by A20 requires TIFA. a WT and A20 KO_1 AGS cells were infected with H. pylori . Total cell lysates were harvested and IP was performed using an antibody against TRAF2 or isotype-matched IgG (IgG). The numbers indicate the band intensities of cIAP1 (normalized to the respective band intensities of TRAF2) of H. pylori -infected samples relative to uninfected control. b WT and TIFA-knockout (TIFA KO ) AGS cells were infected with H. pylori or treated with 30 ng/ml LTα 1 β 2 . Total cell lysates were subjected to IP as in ( a ). c AGS cells were transfected with non-target-specific siRNA (scr) or siRNAs targeting TRAF2 (TRAF2 si ) or cIAP1 (cIAP1 si_8 ) for 48 h prior to infection with H. pylori . Total cell lysates were used for IP with an antibody against TIFA or isotype-matched IgG. d Following incubation of recombinant human A20 and TIFA proteins in vitro, IP was performed using an antibody against TIFA. e as in ( d ) but IP was performed using an antibody against A20 or isotype-matched IgG. f A20 KO_1 AGS cells were infected with H. pylori and IP was performed as in ( c ). IB analyses were performed with TIFA-immunoprecipitates without and with one hour incubation with 100 ng recombinant A20 protein prior to elution of immunoprecipitates. A representative blot of at least two experiments was shown

Article Snippet: The following siRNAs were used: scrambled siRNA (Qiagen, SI03650318); A20 si_5 (Dharmacon, J-009919–05-0005); A20 si_9 (Qiagen, SI05018601); RelB si_E1 (Eurofins, 5’-GACUGCACCGACGGCAUCU-dTT-3’ [ ]); TRAF3 (Santa Cruz Biotechnology, sc-29510); TRAF2 (Santa Cruz Biotechnology, sc-29509); and cIAP1 (Qiagen, SI02654442).

Techniques: Inhibition, Infection, Control, Knock-Out, Transfection, Incubation, Recombinant, In Vitro