anti traf6 rabbit ab  (Cell Signaling Technology Inc)

Journal: Autophagy

Article Title: Picornavirus VP2 protein suppresses innate immunity through selective autophagic degradation of IKBKE/IKKε

doi: 10.1080/15548627.2025.2597460

Figure Lengend Snippet: SVA VP2 protein inhibits the expression of IKBKE and interacts with it. (A) HEK-293T cells were transfected with empty vector or Flag-VP2 plasmids. The cells were lysed at 24 hpt and analyzed by western blotting using the indicated antibodies. (B-D) HEK-293T cells were transfected with 0, 0.25, 0.5 or 1 μg of Flag-VP2 expressing plasmids for 24 h. The expression levels of endogenous IKBKE (B), RIGI (C), or IRF3 (D) were assessed by western blotting. (E) HEK-293T cells were transfected with Flag-VP2 expressing plasmids (0, 0.25, 0.5 or 1 μg) for 24 h. Total rna was extracted from the cells, and IKBKE mRNA levels were quantified by qPCR. (F, G) HEK-293T cells were infected with SVA (MOI = 0.5) for 0, 4, 8 and 12 h respectively, and the protein expression of IKBKE was analyzed by western blotting (F), the mRNA expression of IKBKE was detected by qPCR (G). (H) HEK-293T cells were co-transfected with Flag-VP2 and either Vec, various HA-tagged innate immune molecule-expressing plasmids (MDA5, RIGI, MAVS, TRAF6, TBK1, IRF3, IRF7, IKBKE, and tank) or MYC-tagged TRAF3. At 36 hpt, the cell lysates were subjected to co-IP assay analysis. The immunoprecipitated proteins and whole-cell lysates (WCL) were analyzed by western blotting using the specified antibodies. (I) HEK-293T cells were co-transfected with HA-IKBKE and vector or Flag-VP2 expressing plasmids for 36 h. The cell lysates were immunoprecipitated with anti-HA or control IgG antibodies, and the antigen-antibody complex was subjected to western blotting analysis. (J) HEK-293T cells were mock-infected or infected with SVA at an MOI of 0.5 for 12 h. Cell lysates were then immunoprecipitated with anti-VP2 or control IgG antibodies, the antigen-antibody complex were assessed by western blotting. (K) HEK-293T cells were mock-infected or infected with SVA (MOI = 0.1) for 10 h, after which the colocalization of IKBKE (red) and VP2 (green) was assessed by immunofluorescence assay (IFA). Nuclei were counterstained with DAPI (blue). (l, M) HEK-293T cells were co-transfected with increasing amounts of Flag-VP2 and MYC-IKBKE, along with HA-TBK1 (L) or HA-IRF3 (M) expressing plasmids. At 24 hpt, the cells were treated with SeV for another 12 h. The cells were lysed and immunoprecipitated with anti-MYC antibodies. The immunoprecipitated proteins and WCL were analyzed by western blotting using the specified antibodies.

Article Snippet: The commercial antibodies used in this study include: anti-Flag mouse Ab (Sigma, F1804), anti-MYC mouse Ab (Sigma, M5546), anti-HA mouse Ab (Proteintech, 66,006–2-Ig), anti-RIGI rabbit Ab (Cell Signaling Technology, 3743S), anti-IFIH1/MDA5 rabbit Ab (Abcam, Ab126630 ), anti-MAVS rabbit Ab (Cell Signaling Technology, 3993S), anti-TRAF3 rabbit Ab (Cell Signaling Technology, 4729T), anti-TRAF6 rabbit Ab (Cell Signaling Technology, 67591S), anti-TBK1 rabbit Ab (Cell Signaling Technology, 38066S), anti-IRF3 rabbit Ab (Proteintech, 11,312–1-AP), anti-IRF7 rabbit Ab (Abcam, ab109255), anti-LC3 rabbit Ab (Proteintech, 14,600–1-AP), anti-TUBB/β-tubulin mouse Ab (Abclonal, A12289), anti-IKBKE/IKKε rabbit Ab (Abclonal, A3463), anti-K33 rabbit Ab (Abclonal, A18199), anti-RNF114 rabbit Ab (Abclonal, A10636), anti-CALCOCO2/NDP52 rabbit Ab (Abclonal, A24021), anti-SQSTM1/p62 rabbit Ab (Abclonal, A19700).

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Infection, Co-Immunoprecipitation Assay, Immunoprecipitation, Control, Immunofluorescence

Journal: Autophagy

Article Title: Picornavirus VP2 protein suppresses innate immunity through selective autophagic degradation of IKBKE/IKKε

doi: 10.1080/15548627.2025.2597460

Figure Lengend Snippet: SVA VP2 protein inhibits the expression of IKBKE and interacts with it. (A) HEK-293T cells were transfected with empty vector or Flag-VP2 plasmids. The cells were lysed at 24 hpt and analyzed by western blotting using the indicated antibodies. (B-D) HEK-293T cells were transfected with 0, 0.25, 0.5 or 1 μg of Flag-VP2 expressing plasmids for 24 h. The expression levels of endogenous IKBKE (B), RIGI (C), or IRF3 (D) were assessed by western blotting. (E) HEK-293T cells were transfected with Flag-VP2 expressing plasmids (0, 0.25, 0.5 or 1 μg) for 24 h. Total rna was extracted from the cells, and IKBKE mRNA levels were quantified by qPCR. (F, G) HEK-293T cells were infected with SVA (MOI = 0.5) for 0, 4, 8 and 12 h respectively, and the protein expression of IKBKE was analyzed by western blotting (F), the mRNA expression of IKBKE was detected by qPCR (G). (H) HEK-293T cells were co-transfected with Flag-VP2 and either Vec, various HA-tagged innate immune molecule-expressing plasmids (MDA5, RIGI, MAVS, TRAF6, TBK1, IRF3, IRF7, IKBKE, and tank) or MYC-tagged TRAF3. At 36 hpt, the cell lysates were subjected to co-IP assay analysis. The immunoprecipitated proteins and whole-cell lysates (WCL) were analyzed by western blotting using the specified antibodies. (I) HEK-293T cells were co-transfected with HA-IKBKE and vector or Flag-VP2 expressing plasmids for 36 h. The cell lysates were immunoprecipitated with anti-HA or control IgG antibodies, and the antigen-antibody complex was subjected to western blotting analysis. (J) HEK-293T cells were mock-infected or infected with SVA at an MOI of 0.5 for 12 h. Cell lysates were then immunoprecipitated with anti-VP2 or control IgG antibodies, the antigen-antibody complex were assessed by western blotting. (K) HEK-293T cells were mock-infected or infected with SVA (MOI = 0.1) for 10 h, after which the colocalization of IKBKE (red) and VP2 (green) was assessed by immunofluorescence assay (IFA). Nuclei were counterstained with DAPI (blue). (l, M) HEK-293T cells were co-transfected with increasing amounts of Flag-VP2 and MYC-IKBKE, along with HA-TBK1 (L) or HA-IRF3 (M) expressing plasmids. At 24 hpt, the cells were treated with SeV for another 12 h. The cells were lysed and immunoprecipitated with anti-MYC antibodies. The immunoprecipitated proteins and WCL were analyzed by western blotting using the specified antibodies.

Article Snippet: The commercial antibodies used in this study include: anti-Flag mouse Ab (Sigma, F1804), anti-MYC mouse Ab (Sigma, M5546), anti-HA mouse Ab (Proteintech, 66,006–2-Ig), anti-RIGI rabbit Ab (Cell Signaling Technology, 3743S), anti-IFIH1/MDA5 rabbit Ab (Abcam, Ab126630 ), anti-MAVS rabbit Ab (Cell Signaling Technology, 3993S), anti-TRAF3 rabbit Ab (Cell Signaling Technology, 4729T), anti-TRAF6 rabbit Ab (Cell Signaling Technology, 67591S), anti-TBK1 rabbit Ab (Cell Signaling Technology, 38066S), anti-IRF3 rabbit Ab (Proteintech, 11,312–1-AP), anti-IRF7 rabbit Ab (Abcam, ab109255), anti-LC3 rabbit Ab (Proteintech, 14,600–1-AP), anti-TUBB/β-tubulin mouse Ab (Abclonal, A12289), anti-IKBKE/IKKε rabbit Ab (Abclonal, A3463), anti-K33 rabbit Ab (Abclonal, A18199), anti-RNF114 rabbit Ab (Abclonal, A10636), anti-CALCOCO2/NDP52 rabbit Ab (Abclonal, A24021), anti-SQSTM1/p62 rabbit Ab (Abclonal, A19700).

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Infection, Co-Immunoprecipitation Assay, Immunoprecipitation, Control, Immunofluorescence

Journal: Signal transduction and targeted therapy

Article Title: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) membrane (M) protein inhibits type I and III interferon production by targeting RIG-I/MDA-5 signaling.

doi: 10.1038/s41392-020-00438-7

Figure Lengend Snippet: Fig. 5 The SARS-CoV-2 M protein prevents the formation of the RIG-I–MAVS–TRAF3–TBK1 multiprotein complex. The SARS-CoV-2 M protein inhibits the RIG-I–MAVS (a), MAVS–TBK1 (c), and TRAF3–TBK1 (d) but not MDA-5–MAVS (b) interactions. The HEK293T cells were transfected with the indicated plasmids for 24 h before coimmunoprecipitation with the antibodies against MAVS (a and b) or TBK1 (c and d). The pcDNA6B empty vector was used to balance the total amount of plasmid DNA in the transfection. The input and immunoprecipitates were immunoblotted with the indicated antibodies. Immunoblotting results are representative of two independent experiments. SARS-CoV-2 M protein, SCV2-M

Article Snippet: Antibodies and reagents Rabbit anti-DYKDDDDK tag (D6W5B), rabbit anti-RIG-I (D14G6), rabbit anti-IRF3 (D83B9), rabbit anti-pIRF3 (4D46), rabbit anti-TBK1 (3031S), rabbit anti-pTBK1 (D52C2), and rabbit anti-TRAF3 antibodies (Abs) were obtained from Cell Signaling Technology (USA); the mouse antiMAVS (E-3) and mouse anti-TRAF3 (G-6) antibodies were obtained from Santa Cruz Biotechnology (USA); the mouse anti-actin and rabbit anti-calnexin antibodies were obtained from Proteintech (Wuhan, China); the mouse anti-Flag M2 antibody was obtained from SigmaAldrich (USA); the mouse anti-Myc (9E10) antibody was obtained from Origene (USA); the rabbit anti-GM130 antibody was obtained from Abcam (United Kingdom); the mouse anti-GAPDH antibody (AF0006) was obtained from Beyotime (China); and the mouse anti-HA antibody was obtained from MDL Biotech (China).

Techniques: Transfection, Plasmid Preparation, Western Blot

Journal: Nature Communications

Article Title: TLR7 mediated viral recognition results in focal type I interferon secretion by dendritic cells

doi: 10.1038/s41467-017-01687-x

Figure Lengend Snippet: TLR7 trafficking induces TRAF6–TRAF3 association and IFN-α production in pDC clusters. a , b WT or Arl8b Gt/Gt BM-pDCs were stimulated with 25 μg/mL polyU for the indicated times prior to lysis and immunoprecipitation using Ab against TRAF6 (T6) or goat IgG negative control (C) followed by immunostaining of the indicated signaling molecules. Apparent molecular mass is indicated (left). Immunostaining of TRAF3 in whole-cell lysate is also shown in the lowermost panel ( a ). Immunoprecipitation of the same cell lysates as in a but using Ab against TRAF3 (T3) or rabbit IgG negative control (C) prior to immunostaining ( b ). c WT and Arl8b Gt/Gt BM-pDCs were either left unstimulated (US) or stimulated with 25 μg/mL polyU (pU) for 3 h. Cells were then stained with Abs against TLR7 and phosphorylated mTOR (p-mTOR) as indicated. Nuclei were stained with DAPI. Higher magnification images of boxed regions are shown in the insets. Statistical determination of TLR7 co-localization with p-mTOR is shown in the panel below ( n > 24). *** P < 0.001, * P < 0.05 (unpaired two-tailed t -test). Scale bar, 5 μm. d , e WT BM-pDCs were either left unstimulated (US) or activated (pU) with 25 μg/mL polyU for 6–10 h. Cells were stained with an Ab against Interferon α (IFNα). Scale bar, 10 μm (left and middle), 5 μm (right). In IFN-α-producing pDCs, the percentages of clustered and isolated pDCs were determined ( n = 324). Also, in isolated pDCs, the percentages of IFN-α-positive and -negative pDCs were determined ( n = 600). In pDC doublets, the percentages of pDCs with IFN-α in the cell–cell contact, ER/Golgi, or miscellaneous area are shown ( n = 276)

Article Snippet: Ab obtained from commercial sources included rat anti-α-tubulin (NOVUS Biologicals), rat anti-mouse IFN-α (Hycult Biotech), rat anti-CD11a (M17/4), CD102 (3C4), CD18 (M18/2), and CD54 (YN1/1.7.4) (Biolegend), rabbit anti-Rab5, Rab7a, PKD, p-PKD (Ser916), p-PKD (Ser744/748), IKKα, MyD88, NF-κB p65, p-NF-κB p65 (Ser536), p-S6 (Ser240/244), S6 ribosomal protein, p-S6 kinase (Thr389), S6 kinase, Rap1A/Rap1B, p-mTOR, mTOR, and RAPTOR (Cell Signaling), rabbit anti-IRF7 (Bio-Rad AbD Serotec), rabbit anti-calnexin and TRAF6 (Abcam), goat anti-TRAF6 and rabbit anti-TRAF3 Abs (Santa Cruz), mouse anti-actin (Sigma-Aldrich), mouse anti-AP3 δ (DSHB).

Techniques: Lysis, Immunoprecipitation, Negative Control, Immunostaining, Staining, Two Tailed Test, Isolation

Journal: PLoS ONE

Article Title: Roles of the Kinase TAK1 in TRAF6-Dependent Signaling by CD40 and Its Oncogenic Viral Mimic, LMP1

doi: 10.1371/journal.pone.0042478

Figure Lengend Snippet: A) A20.TRAF3 +/+ or A20.TRAF3 −/− mouse B cells were stimulated with anti-mCD40 Ab for 0, 5, 10, or 30 min. Western blots show pTAK1 and TRAF3, with actin as a loading control. B) Similar to panel A , except that A20.TRAF6 +/+ or A20.TRAF6 −/− mouse B cells were used. Western blots show pTAK1 and TRAF6, with actin as a loading control. These images were part of the same membrane, but the middle lanes were omitted here for clarity. Data shown are representative of at least four independent experiments.

Article Snippet: The following primary antibodies were used for Western blotting: rabbit anti-phospho-JNK Ab, rabbit anti-phospho-TAK1 Ab, rabbit anti-phospho-IκBα Ab, rabbit anti-total IκBα Ab, and rabbit anti-phospho-c-jun Ab (Cell Signaling Technology, Danvers, MA), rabbit anti-total JNK Ab and rabbit anti-TRAF3 Ab (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-actin Ab (Millipore, Billerica, MA or Sigma-Aldrich), rabbit anti-TRAF2 Ab, chicken anti-TRAF6 Ab, and hamster anti-TRAF6 Ab (Medical and Biological Laboratories, Japan), and S12 (mouse anti-LMP1 IgG mAb; produced from a hybridoma that was a gift from Dr. Fred Wang, Harvard University, Cambridge, MA).

Techniques: Western Blot, Control, Membrane

Journal: PLoS ONE

Article Title: Roles of the Kinase TAK1 in TRAF6-Dependent Signaling by CD40 and Its Oncogenic Viral Mimic, LMP1

doi: 10.1371/journal.pone.0042478

Figure Lengend Snippet: A) CH12.TRAF3 +/+ or CH12.TRAF3 −/− mouse B cells expressing hCD40 were stimulated with control (−) or anti-hCD40 (+) Ab-coated beads for 10 min. hCD40 was immunoprecipitated using the same beads. B) Quantitation by luminescence imaging of the results of 3 independent experiments, mean values ± SEM. *p<0.05.

Article Snippet: The following primary antibodies were used for Western blotting: rabbit anti-phospho-JNK Ab, rabbit anti-phospho-TAK1 Ab, rabbit anti-phospho-IκBα Ab, rabbit anti-total IκBα Ab, and rabbit anti-phospho-c-jun Ab (Cell Signaling Technology, Danvers, MA), rabbit anti-total JNK Ab and rabbit anti-TRAF3 Ab (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-actin Ab (Millipore, Billerica, MA or Sigma-Aldrich), rabbit anti-TRAF2 Ab, chicken anti-TRAF6 Ab, and hamster anti-TRAF6 Ab (Medical and Biological Laboratories, Japan), and S12 (mouse anti-LMP1 IgG mAb; produced from a hybridoma that was a gift from Dr. Fred Wang, Harvard University, Cambridge, MA).

Techniques: Expressing, Control, Immunoprecipitation, Quantitation Assay, Imaging