Recombinant Human TRAF3, GST-tagged

• Cat.No.: TRAF3-3384H
• Product Overview: Recombinant Human TRAF3 protein, fused to GST-tag, was expressed in E.coli and purified by GSH-sepharose.

Specification

• Source: E.coli
• Species: Human
• Tag: GST
• Protein length: 6-351aa
• Storage: The protein is stored in PBS buffer at -20℃. Avoid repeated freezing and thawing cycles.
• Storage Buffer: 1M PBS (58mM Na2HPO4,17mM NaH2PO4, 68mM NaCl, pH8. ) added with 100mM GSH and 1% Triton X-100,15%glycerol.

Gene Information

• Gene Name: TRAF3 TNF receptor-associated factor 3 [ Homo sapiens ]
• Official Symbol: TRAF3
• Synonyms: TRAF3; TNF receptor-associated factor 3; CAP 1; CD40bp; CRAF1; LAP1; CD40 binding protein; CD40 associated protein 1; LMP1-associated protein 1; CD40 receptor associated factor 1; CAP-1
• Gene ID: 7187
• mRNA Refseq: NM_001199427
• Protein Refseq: NP_001186356
• MIM: 601896
• UniProt ID: Q13114
• Chromosome Location: 14q32.32
• Pathway: Apoptosis, organism-specific biosystem; CD40/CD40L signaling, organism-specific biosystem; Hepatitis C, organism-specific biosystem; Hepatitis C, conserved biosystem; Herpes simplex infection, organism-specific biosystem; Herpes simplex infection, conserv
• Function: ligase activity; metal ion binding; protein binding; signal transducer activity; ubiquitin-protein ligase activity; zinc ion binding

Citation

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

Journal: Nature Communications    PubMed ID: 33608556    Data: 2021/2/19

Authors: Peng Gao, Xianwei Ma, Huazhang An

Article Snippet:LTA (L2515) and Protein G Sepharose (P3296) were from Sigma.LTA (L2515) and Protein G Sepharose (P3296) were from Sigma.. GST-TRAF3 (3384H) was from Creative BioMart.. Recombinant Nedd4l (TP327866) and recombinant TRAF3 (TP318682) were from Origene.Recombinant Nedd4l (TP327866) and recombinant TRAF3 (TP318682) were from Origene.

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.

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.

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.