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: 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: mBio

Article Title: Lumpy skin disease virus LSDV001 protein positively regulates inflammatory response by promoting assembly of the TAK1-TAB2/3 complex

doi: 10.1128/mbio.01677-25

Figure Lengend Snippet: LSDV001 promotes assembly of the TAK1-TAB2/3 complex. ( A ) Effects of LSDV001 on the association of TAK1 with TABs. HEK293 cells (1 × 10 7 ) were transfected with the indicated plasmids for 24 hours, followed by coimmunoprecipitation and immunoblot analysis. ( B and C ) Effects of LSDV001 on the endogenous association of TRAF6 or TRAF2 with TAK1 and TAB2/3, as well as TAB2/3 with TAK1 following IL-1β or TNFα stimulation. HEK293 cells (2 × 10 7 ) were transfected with an empty vector or LSDV001-Flag plasmid for 24 hours. The cells were left untreated or treated with IL-1β or TNFα for 15 minutes before cells were harvested for immunoprecipitation with control IgG or antibodies against TAK1, TAB2, TRAF6, or TRAF2. The lysates and immunoprecipitates were subjected to immunoblot analysis with the indicated antibodies.

Article Snippet: Mouse monoclonal antibodies against HA (66006, Proteintech); rabbit monoclonal antibodies against HA (H6908, Sigma); Mouse monoclonal antibodies against Flag (F3165, Sigma); HRP-Flag (ZB15939, Servicebio); IgG (I5381 and I5006, Sigma); p-TAK1 (Thr184/187) (4508, Cell Signaling Technology), TAK1, TAB3, TRAF2, and TRAF6 (A19077, A19824, A19129, and A19686, ABclonal); TAB2 (3745, CST); β-actin and p65 (sc-47778 and 71675; Santa Cruz Biotechnology); p-p65(S536) and p-IKKα (Ser176)/IKKβ(Ser177) (3033 and 2078, Cell Signaling Technology); IκBα and p-IκBα (9242 and 9246, Cell Signaling Technology), anti-mouse IgG (H + L), F(ab′)2 Fragment (Alexa Fluor 594 Conjugate) (8890, Cell Signaling Technology); Alexa Fluor 488 goat anti-rabbit IgG (H + L) (A11008, Invitrogen) were purchased from the indicated manufacturers.

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

Journal: Cell Death & Disease

Article Title: Dual role of DR5 in death and survival signaling leads to TRAIL resistance in cancer cells

doi: 10.1038/cddis.2017.423

Figure Lengend Snippet: TRAIL induces the assembly of apoptotic and non-apoptotic signaling complex(es). ( a ) Surface levels of Death Receptor 5 (anti-DR5-488), Death Receptor 4 (anti-DR4 PE), Decoy Receptor 1 (anti-DcR1 PE) and Decoy Receptor 2 (anti-DcR2-488) in BJELR cells. Surface levels of Death Receptor 4 in Hela cells and surface level of Decoy Receptor 1 in BJ cells were used as a positive control for DR4 and DcR1 labeling, respectively. Isotypic IgG1PE or Alexa 488 or IgG2B Alexa 488 labeling was used as a control for background fluorescence. ( b ) Western blots assessing the co-immunoprecipitation of indicated proteins (canonical DISC components) with Death Receptor 5 (immunoprecipitation target; DR5 IP), Death Receptor 4 (immunoprecipitation target; DR4 IP) or Decoy Receptor 2 (immunoprecipitation target; DcR2 IP) from whole-cell lysates obtained from naïve cells (−) or cells treated with TRAIL for 30 min (+). NF, not detected. ( c ) Western blots assessing the co-immunoprecipitation of RIPK1 and TRAF2 as well as canonical DISC components (internal control) with Death Receptor 5 (DR5 IP) from whole-cell lysates obtained from naïve cells (−) or cells treated with 1 μ g/ml TRAIL for 30 min (+). ( d ) Western blots assessing the co-immunoprecipitation of RIPK1 and TRAF2 as well as canonical DISC components (internal control) with caspase-8 (immunoprecipitation target; C8 IP) from whole-cell lysates obtained from naïve cells (−) or cells treated with TRAIL for 30 min (+). ( b – d ) Depicted images correspond to one representative experiment out of three independent biological replicates. Immunoprecipitation using isotypic IgG1 (IgG) was used as a background control

Article Snippet: Antibodies for immunoprecipitation of DR5 (AF631), DR4 (AF347), DcR2 (AF633), TRAF2 (AF3277) and normal goat IgG (AB-108-C) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Positive Control, Labeling, Control, Fluorescence, Western Blot, Immunoprecipitation

Journal: Cell Death & Disease

Article Title: Dual role of DR5 in death and survival signaling leads to TRAIL resistance in cancer cells

doi: 10.1038/cddis.2017.423

Figure Lengend Snippet: Role of TRAIL receptors and TRAIL-receptor-binding proteins in the triggering of apoptosis. ( a ) Percentage of cells with high levels of cleaved PARP in populations transfected with pooled siRNAs targeting DR4 (DR4), DR5 (DR5), DcR2 (DcR2) mRNAs or non-targeting scramble siRNAs (scr) further left either untreated (control) or challenged with TRAIL (6 h). ( b ) Percentage of cells with high levels of cleaved PARP in naïve (control) or TRAIL-challenged (6 h) DR5 knockout (DR5 KO) or DcR2 knockout (DcR2 KO) cells as compared with controls (ctrol). ( c ) Contour plot for cleaved PARP immunolabelling in DcR2 knockout (DcR2 KO) cells either unchallenged (control) or treated 6 h with TRAIL (TRAIL). Subpopulations displaying low (survivors, '1') and high (apoptotic, '2') levels of PARP cleavage in treated samples are depicted. Image shows one representative experiment out of three independent biological replicates. ( d ) Percentage of cells with high levels of cleaved PARP in control and DcR2 KO naïve cells (control) or control and DcR2 KO cells challenged with TRAIL for 16 h (TRAIL). ( e ) Percentage of cells with high levels of cleaved PARP in cells transfected with siRNAs targeting FADD (FADD) , caspase-8 (casp-8), RIPK1 (RIPK1), TRAF2 (TRAF2) or cFlip (cFlip) mRNAs or non-targeting scramble siRNAs (scr) further left either untreated (control) or challenged for 6 h with TRAIL (TRAIL). ( f ) Percentage of cells with high levels of cleaved PARP in populations transfected with siRNAs targeting RIPK1 (RIPK1) , TRAF2 (TRAF2) or RIPK1 and TRAF2 (RIPK1+TRAF2) mRNAs or non-targeting scramble siRNAs at concentrations corresponding to single (30 nM; scr 30) or double (60 nM; scr 60) transfections, further left either untreated (control) or challenged during 6 h with TRAIL (TRAIL). Results obtained in conditions of pan-caspase inhibition (control+ zVAD and TRAIL+zVAD) as compared with basal conditions of cell response (control and TRAIL) are shown. ( g ) Percentage of cells with high levels of cleaved PARP in cells transfected with siRNAs targeting RIPK1 (RIPK1) , TRAF2 (TRAF2) , DR5 (DR5), RIPK1 and DR5 (RIPK1+DR5), TRAF2 and DR5 (TRAF2+DR5) mRNAs or non-targeting scramble siRNAs ('scr 30' and 'scr 60') further left either untreated (control) or challenged 6 h with TRAIL (TRAIL). ( a , b and d – g ) Mean±standard deviation (S.D.) from three independent biological replicates is shown. *** P- value <0.0005, ** P- value <0.005, * P- value <0.05. NS, not significant

Article Snippet: Antibodies for immunoprecipitation of DR5 (AF631), DR4 (AF347), DcR2 (AF633), TRAF2 (AF3277) and normal goat IgG (AB-108-C) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Binding Assay, Transfection, Control, Knock-Out, Inhibition, Standard Deviation

Journal: Cell Death & Disease

Article Title: Dual role of DR5 in death and survival signaling leads to TRAIL resistance in cancer cells

doi: 10.1038/cddis.2017.423

Figure Lengend Snippet: Role of TRAILRs-binding proteins in the triggering of non-apoptotic and pro-survival pathways. ( a ) Western blots displaying total and phosphorylated protein levels of Erk1/2, Akt and I κ B α in cells transfected with siRNAs targeting RIPK1 (siRIPK1), TRAF2 (siTRAF2) mRNAs or non-targeting scramble siRNAs (scr) further left either untreated (0) or challenged with TRAIL. ( b ) Western blots displaying total and phosphorylated protein levels of p38 in BJELR cells transfected with siRNAs targeting TRAF2 (siTRAF2) mRNA or non-targeting scramble siRNAs (scr) further left either untreated (0) or challenged with TRAIL. ( c ) Western blots displaying total and phosphorylated protein levels of p38 in cells transfected with siRNAs targeting RIPK1 (siRIPK1) mRNA or non-targeting scramble siRNAs (scr) further left either untreated (0) or challenged with TRAIL. ( d and e ) Western blots displaying total and phosphorylated protein levels of p38 and I κ B α ( d ), Erk1/2 and Akt ( e ) in cells transfected with siRNAs targeting cFlip (sicFlip) mRNA or non-targeting scramble siRNAs (scr) further left either untreated (0) or challenged with TRAIL. Uncropped image for ( e ) is depicted as . ( f ) Western blots displaying total and phosphorylated protein levels of I κ B α and Akt in cells transfected with siRNAs targeting caspase-8 (sicasp8) mRNA or non-targeting scramble siRNAs (scr) further left either untreated (0) or challenged with TRAIL. ( g ) Western blots displaying total and phosphorylated protein levels of I κ B α and Akt in cells transfected with siRNAs targeting FADD (siFADD) mRNA or non-targeting scramble siRNAs (scr) further left either untreated (0) or challenged with TRAIL. ( h ) Western blots displaying total and phosphorylated protein levels of Erk1/2 and p38 in BJELR cells transfected with siRNAs targeting caspase-8 (sicasp8), FADD (siFADD) mRNAs or non-targeting scramble siRNAs (scr) further left either untreated (0) or challenged with TRAIL. ( i ) Western blots displaying total and phosphorylated protein levels of Erk1/2 in BJELR cells transfected with siRNAs targeting FADD (siFADD) mRNA or non-targeting scramble siRNAs (scr) further left either untreated (0) or challenged for the indicated time points with TRAIL. ( a – i ) Depicted images correspond to one representative experiment out of three independent biological replicates. α -Tubulin, loading control

Article Snippet: Antibodies for immunoprecipitation of DR5 (AF631), DR4 (AF347), DcR2 (AF633), TRAF2 (AF3277) and normal goat IgG (AB-108-C) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Binding Assay, Western Blot, Transfection, Control

Journal: Cell Death & Disease

Article Title: Dual role of DR5 in death and survival signaling leads to TRAIL resistance in cancer cells

doi: 10.1038/cddis.2017.423

Figure Lengend Snippet: Apoptotic and pro-survival TRAIL-induced signaling complex(es) are assembled at the plasma membrane. ( a ) Western blots assessing the co-immunoprecipitation of canonical DISC components (DR4, DcR2, FADD, caspase-8, cFlip) as well as RIPK1 and TRAF2 with Death Receptor 5 (DR5 immunoprecipitation: DR5 IP). ( b and c ) Western blots assessing the co-immunoprecipitation of DR5, DR4, DcR2, FADD, cFlip and TRAF2 ( b ) as well as caspase-8 ( c ) with RIPK1 (RIPK1 immunoprecipitation: RIPK1 IP). ( d ) Western blots assessing the co-immunoprecipitation of canonical DISC components (DR4, DR5, DcR2, FADD, caspase-8, cFlip) as well as RIPK1 with TRAF2 (TRAF2 immunoprecipitation: TRAF2 IP). ( e ) Western blots assessing the co-immunoprecipitation of DR5, RIPK1 and TRAF2 with FADD (FADD immunoprecipitation: FADD IP). ( a–e ) Immunoprecipitations were performed from the purified plasma membrane (plasma membrane) obtained from either naïve cells (−) or cells treated with TRAIL (30 min '+' unless indicated otherwise in c ). Immunoprecipitation using isotypic IgG1 (IgG) was used as a background control. Protein levels of indicated proteins at the plasma membrane are depicted as 'inputs'. Same blots are depicted as inputs for ( d ) and ( e ) as TRAF2 and FADD immunoprecipitation experiments were performed in parallel using the same protein input. Depicted images correspond to one representative experiment out of at least two independent biological replicates. N-Cadherin (N-Cdh) is shown as a plasma membrane marker. Controls for plasma membrane purification are depicted as . ND, not detected

Article Snippet: Antibodies for immunoprecipitation of DR5 (AF631), DR4 (AF347), DcR2 (AF633), TRAF2 (AF3277) and normal goat IgG (AB-108-C) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Clinical Proteomics, Membrane, Western Blot, Immunoprecipitation, Purification, Control, Marker

Journal: Cell Death & Disease

Article Title: Dual role of DR5 in death and survival signaling leads to TRAIL resistance in cancer cells

doi: 10.1038/cddis.2017.423

Figure Lengend Snippet: Role of TRAIL receptors in the formation of TRAIL-induced signaling platforms. ( a ) Western blots assessing the co-immunoprecipitation of canonical DISC components (DR4, DcR2, FADD, caspase-8, cFlip) with Death receptor 5 (DR5 immunoprecipitation: DR5 IP) in plasma membrane fractions obtained from DcR2 knockout (DcR2 KO) and control BJELR cells either left untreated (−) or challenged with TRAIL (30 min '+'). ( b ) Western blots assessing the co-immunoprecipitation of DR5 with RIPK1 (RIPK1 immunoprecipitation: RIPK1 IP) or TRAF2 (TRAF2 immunoprecipitation: TRAF2 IP) in DcR2 knockout (DcR2 KO) and control BJELR cells either left untreated (−) or challenged with TRAIL (30 min '+'). ( c ) Western blots assessing the co-immunoprecipitation of canonical DISC components (DR5, DcR2, FADD, caspase-8) with Death receptor 4 (DR4 immunoprecipitation: DR4 IP) in plasma membrane fractions obtained from DR5 knockout (DR5 KO) and control BJELR cells either left untreated (−) or challenged with TRAIL (30 min '+'). ( d ) Western blots assessing the co-immunoprecipitation of canonical DISC components (DR5, DR4, DcR2, FADD) with RIPK1 (RIPK1 immunoprecipitation: RIPK1 IP) or TRAF2 (TRAF2 immunoprecipitation: TRAF2 IP) in DR5 knockout (DR5 KO) and control BJELR cells either left untreated (−) or challenged with TRAIL (30 min '+'). ( e ) Western blots assessing the co-immunoprecipitation of canonical DISC components (DR5, DR4, DcR2, FADD, cFlip) with caspase-8 (caspase-8 immunoprecipitation: C8 IP) in whole-cell lysates from DR5 knockout (DR5 KO) and control BJELR cells either left untreated (−) or challenged with TRAIL (30 min '+'). ( a – c and e ) Controls for plasma membrane purification are depicted as . ND, not detected; NA, not analyzed

Article Snippet: Antibodies for immunoprecipitation of DR5 (AF631), DR4 (AF347), DcR2 (AF633), TRAF2 (AF3277) and normal goat IgG (AB-108-C) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, Immunoprecipitation, Clinical Proteomics, Membrane, Knock-Out, Control, Purification

Journal: Nature communications

Article Title: NEDD4 ubiquitinates TRAF3 to promote CD40-mediated AKT activation.

doi: 10.1038/ncomms5513

Figure Lengend Snippet: Figure 2 | NEDD4 promotes TRAF3 K63-linked ubiquitination upon CD40 engagement. (a) 293T cells were transfected with Myc-tagged Ubiquitin (Myc-Ub), HA-TRAF2, HA-TRAF3 and Flag-NEDD4 as indicated. Cell lysates were immunoprecipitated (IP) with anti-HA. Immune complexes were gel-separated and immunoblotted with anti-HA. (b) 293T cells were transfected with Myc-Ub, HA-TRAF3, increasing doses of Flag-NEDD4 or Flag-NEDD4 ligase-dead mutant (Mut) as indicated. Cell lysates were immunoprecipitated (IP) with anti-HA and the immune complexes were immunoblotted with anti-HA. (c) A20 cells transfected with control or NEDD4 short interfering RNAs were stimulated with CD40L. At the indicated times, cell lysates were prepared and immunoblotted with antibodies to TRAF3, NEDD4 and a-tubulin. (d) 293Tcells were transfected with Myc-Ub, HA-TRAF3 and Flag-NEDD4 as indicated. After 36 h, cell lysates were prepared and immunoprecipitated with anti-HA, stringently washed and analysed by immunoblotting with linkage-specific antibodies recognizing K48- and K63-linked polyubiquitin. (e) A20 cells transduced with lentiviruses containing control or NEDD4 shRNAs were stimulated with CD40L. At the indicated times, cell lysates were prepared and immunoprecipitated with anti-TRAF3 and the immune complexes were stringently washed and immunoblotted with linkage-specific antibodies recognizing K48- and K63-linked polyubiquitin. Data are representative of at least three independent experiments.

Article Snippet: Antibodies specific to phosph-p38 (Thr180/Tyr182) (9211, Cell Signaling Technology); CD40 (sc-975, Santa Cruz Biotechnology; Supplementary Fig. 2a); NEDD4 (2740, Cell Signaling Technology); TRAF3 (sc-947 and sc-6933, Santa Cruz Biotechnology); FOXO1 (1874-1, EPITOMICS), PhosphoFOXO1(Ser256) (9461, Cell Signaling Technology); FOXO3a (2497, Cell Signaling Technology); phosph-FOXO3a (ser253) (13129, Cell Signaling Technology); HAprobe Antibody (sc-7392 and sc-805, Santa Cruz Biotechnology); purified Rat AntiMouse CD43 (Ly-48, Leukosialin) Monoclonal Antibody (553268, BD Pharmingen); rabbit IgG (sc-2027, Santa Cruz Biotechnology), p85 (06-496, Merck Millipore, dilution 1:5,000); a-tubulin (T5168, Adam Equipment, dilution 1:10,000); JNK (9252, Cell Signaling Technology); phosph-JNK (Thr183/Tyr185) (9251, Cell Signaling Technology); NF-kB p65 (C-20) (sc-372, Santa Cruz, dilution 1:5,000); Phospho-IkBa (Ser32) (2859, Cell Signaling Technology); IkBa (C-21) (SC-371, Santa Cruz); TRAF2 (Clone 324522) (MAB3277, R&D Systems); P100/P52 (4882, Cell Signaling Technology); Phospho-AKT(Ser473) (9271, Cell Signaling Technology); AKT (9272, Cell Signaling Technology); Recombinant epidermal growth factor (236-EG-01M, R&D systems); Wortmannin (681675, Calbiochem); lipopolysaccharide (L2880, Sigma); Syndecan-1 (142509, BioLegend); B220 (103207, BioLegend).

Techniques: Ubiquitin Proteomics, Transfection, Immunoprecipitation, Mutagenesis, Control, Western Blot, Transduction

Journal: Cardiovascular Research

Article Title: The tumour suppressor Ras-association domain family protein 1A (RASSF1A) regulates TNF-α signalling in cardiomyocytes

doi: 10.1093/cvr/cvu111

Figure Lengend Snippet: RASSF1A plays a key role in the recruitment of TRAF2 and TRADD to the TNFRC. ( A ) Isolated adult cardiomyocytes from RASSF1A −/− mice and WT littermates were treated with 10 ng/mL of TNF-α for 30 min. Immunoprecipitation analysis indicated a marked reduction of TRADD and ( B ) TRAF2 co-precipitation with TNFR1 in RASSF1A −/− cardiomyocytes compared with WT ( n = 4 independent animals). ( C ) Immunoprecipitation analysis of neonatal rat cardiomyocytes (NRCMs) treated with 10 ng/mL of TNF-α. Total protein lysates were precipitated using antibodies as indicated in the figure. Western blot analyses showed that RASSF1A co-precipitated with TNFR1, TRADD, and TRAF2. However, RASSF1A did not interact with TNFR2. ( D ) Overexpression of RASSF1A deletion constructs in NRCM using adenoviral constructs. Expression was detected using an anti-Flag antibody and GAPDH as a loading control in total protein lysates. ( E ) Immunoprecipitation analysis showed that only RASSF1A-ΔN was co-precipitated with TRAF2. Both the ΔC and ΔC+RA constructs did not co-precipitate with TRAF2, suggesting that the C-terminal domain of RASSF1A was responsible for binding with TRAF2. ( F ) Similarly, the C-terminal region was also important in mediating interaction with TRADD ( n = 3 independent experiments).

Article Snippet: Plasmid containing human TNFR-associated factor 2 (TRAF2) cDNA was a gift of Dr John Kyriakis (Boston, MA, USA; Addgene plasmid #21586).

Techniques: Isolation, Immunoprecipitation, Western Blot, Over Expression, Construct, Expressing, Control, Binding Assay

Journal: Cardiovascular Research

Article Title: The tumour suppressor Ras-association domain family protein 1A (RASSF1A) regulates TNF-α signalling in cardiomyocytes

doi: 10.1093/cvr/cvu111

Figure Lengend Snippet: RASSF1A gene knockdown using shRNA reduces the formation of TNFRC and the activation of NFκB pathway. ( A ) Western blot analysis showed ablation of RASSF1A expression in NRCM treated with Ad-shRASSF1A. ( B ) Immunoprecipitation experiments suggested a significant reduction in the interaction between TNFR1–TRAF2 and TNFR1–TRADD in NRCM treated with Ad-shRASSF1A. All cells were stimulated with 10 ng/mL of TNF-α. ( C ) Activation of the NFκB pathway was assessed using adenoviral-driven NFκB-luciferase construct. RASSF1A gene inactivation significantly reduced NFκB activation in response to TNF-α induction. This phenotype was restored by overexpression of human RASSF1A (* P < 0.05, n = 3 independent experiments).

Article Snippet: Plasmid containing human TNFR-associated factor 2 (TRAF2) cDNA was a gift of Dr John Kyriakis (Boston, MA, USA; Addgene plasmid #21586).

Techniques: Knockdown, shRNA, Activation Assay, Western Blot, Expressing, Immunoprecipitation, Luciferase, Construct, Over Expression

Journal: Journal of cell science

Article Title: TNFα induced noncanonical NF-κB activation is attenuated by RIP1 through stabilization of TRAF2.

doi: 10.1242/jcs.075770

Figure Lengend Snippet: Fig. 1. TNFinduces activation of the noncanonical but not canonical NF- B pathway in RIP1–/– MEFs. (A)Western blots showing p100 processing in TNF-treated wild-type and RIP1–/– MEFs. Cells were treated with TNF(30 ng/ml) for indicated times and cell lysates were applied for western blot with indicated antibodies. (B)(Top) Electrophoretic mobility shift assay (EMSA) in nuclear cell lysates from wild-type, TRAF2–/– and RIP1–/– MEFs treated with TNFshowing binding to NF-B probe. (Bottom) As a control, an anti-Sp-1 antibody was used in western blot of the same nuclear lysates. (C)Western blots of lysates from TNF-treated wild-type and RIP1–/– MEFs showing reduced IBdegradation in RIP1–/– MEFs. (D)Western blots from RIP1–/–

Article Snippet: Recombinant murine and human TNF, mouse recombinant TRAIL, human recombinant LIGHT, agonistic (AF-425-PB) and blocking (MAB430) TNFR1 antibodies, TRAF2 antibody and zVAD were purchased from R&D Systems.

Techniques: Activation Assay, Western Blot, Electrophoretic Mobility Shift Assay, Binding Assay, Control

Journal: Journal of cell science

Article Title: TNFα induced noncanonical NF-κB activation is attenuated by RIP1 through stabilization of TRAF2.

doi: 10.1242/jcs.075770

Figure Lengend Snippet: Fig. 2. Deficiency of RIP1 leads to p100 processing and TRAF2 degradation in response to TNF. (A)Western blots of lysates from TNF- treated wild-type, RIP1–/– and TRAF2–/– MEFs showing TRAF2 degradation and p52 generation in RIP1–/– MEFs. (B)Western blots of lysates from short time course of TNF-treated RIP1–/– MEFs showing a correlation between TRAF2 degradation and p52 generation. (C)Western blots of lysates from wild-type and RIP1–/– MEFs showing TRAF2 degradation in TNF-treated RIP1–/– MEFs cells, but lack of TRAF3 degradation. (D)Western blots of lysates from wild-type MEFs transfected with non-target control siRNA (NC) or RIP1 siRNA and treated with TNFfor indicated times, showing degradation of TRAF2 in RIP1-knockdown wild-type MEFs. (E)Western blot showing protein expression levels of RIP1 in wild-type, RIP1–/– and RIP1–/–

Article Snippet: Recombinant murine and human TNF, mouse recombinant TRAIL, human recombinant LIGHT, agonistic (AF-425-PB) and blocking (MAB430) TNFR1 antibodies, TRAF2 antibody and zVAD were purchased from R&D Systems.

Techniques: Western Blot, Transfection, Control, Knockdown, Expressing

Journal: Journal of cell science

Article Title: TNFα induced noncanonical NF-κB activation is attenuated by RIP1 through stabilization of TRAF2.

doi: 10.1242/jcs.075770

Figure Lengend Snippet: Fig. 4. Degradation of TRAF2 induces NIK accumulation and phosphorylation of IKK. (A)Western blots of NIK and TRAF2 in TNF (30 ng/ml)-treated wild-type, RIP1–/– and TRAF2–/– MEFs for the indicated times. (B)Western blots of longer time course showing NIK accumulation and IKKphosphorylation in response to TNFin RIP1–/– MEFs.

Article Snippet: Recombinant murine and human TNF, mouse recombinant TRAIL, human recombinant LIGHT, agonistic (AF-425-PB) and blocking (MAB430) TNFR1 antibodies, TRAF2 antibody and zVAD were purchased from R&D Systems.

Techniques: Phospho-proteomics, Western Blot

Journal: Journal of cell science

Article Title: TNFα induced noncanonical NF-κB activation is attenuated by RIP1 through stabilization of TRAF2.

doi: 10.1242/jcs.075770

Figure Lengend Snippet: Fig. 5. RIP1 reduces the recruitment of TRAF2 to TNFR1 and TRADD. (A,B) Western blots of immunoprecipitates in TNF(30 ng/ml)-treated wild- type or RIP1–/– MEFs immunoprecipitated with (A) anti-TNFR1 antibody or (B) anti-TRADD antibody. Input: 1% of total lysates.

Article Snippet: Recombinant murine and human TNF, mouse recombinant TRAIL, human recombinant LIGHT, agonistic (AF-425-PB) and blocking (MAB430) TNFR1 antibodies, TRAF2 antibody and zVAD were purchased from R&D Systems.

Techniques: Western Blot, Immunoprecipitation

Journal: Journal of cell science

Article Title: TNFα induced noncanonical NF-κB activation is attenuated by RIP1 through stabilization of TRAF2.

doi: 10.1242/jcs.075770

Figure Lengend Snippet: Fig. 7. Inhibition of noncanonical NF-kB pathway causes TNF-induced cell death in RIP1–/– MEFs. (A)Western blots of lysates from RIP1–/– MEFs transfected with non-target control siRNA or NIK siRNA and treated with TNF(2 hours) showing that TRAF2 continues to be degraded in NIK- knockdown RIP1–/– MEFs. (B,C)Viability assays of RIP1–/– MEFs transfected with NIK siRNA or non-targeted control (NC) siRNA and treated with TNF showing the effect of NIK knockdown on TNF-induced apoptosis in RIP1–/–

Article Snippet: Recombinant murine and human TNF, mouse recombinant TRAIL, human recombinant LIGHT, agonistic (AF-425-PB) and blocking (MAB430) TNFR1 antibodies, TRAF2 antibody and zVAD were purchased from R&D Systems.

Techniques: Inhibition, Western Blot, Transfection, Control, Knockdown