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: The Journal of Neuroscience

Article Title: The Ubiquitin E3 Ligase TRAF6 Exacerbates Ischemic Stroke by Ubiquitinating and Activating Rac1

doi: 10.1523/JNEUROSCI.1751-17.2017

Figure Lengend Snippet: Antibodies used for immunofluorescence

Article Snippet: The protein bands were detected using a Bio-Rad imaging system and specific protein expression levels were normalized to GAPDH protein. table ft1 table-wrap mode="anchored" t5 Table 3. caption a7 Antibody Manufacturer Catalog # Sources of species RRID TRAF6 Abcam Ab40675 Rabbit AB_778573 Bax CST 2772 Rabbit AB_329921 Bcl-2(50E 3 ) CST 2870 Rabbit AB_2290370 Cleaved Caspase3(Asp175) CST 9661 Rabbit AB_2341188 Caspase-3 CST 9662 Rabbit AB_331439 p-IkBa(ser32/36) CST 9246L Mouse AB_2267145 IkBa(L35A5) CST 4814 Mouse AB_390781 P-Ikkβ(Y199) Abcam Ab59195 Rabbit AB_943810 IKKβ CST 8943S Rabbit AB_11024092 P-NF-kB p65(Ser536) CST 3033 Rabbit AB_331284 p65 CST 4764 Rabbit AB_823578 P47-phox(P366) Bioworld BS3261 Rabbit AB_1662897 NOXA2/P67 phox Abcam {"type":"entrez-nucleotide","attrs":{"text":"Ab109366","term_id":"37572651","term_text":"AB109366"}} Ab109366 Rabbit AB_10860640 SD1(SOD1) Abcam Ab13498 Rabbit AB_300402 Heme Oxygenase 1(HO-1) Abcam Ab13243 Rabbit AB_299790 p-Rac1/cdc42(Ser71) CST 2461S Rabbit AB_2300703 Rac1/2/3 CST 2467 Rabbit AB_2176150 HA Sigma-Aldrich H6908 Rabbit AB_260070 Flag Sigma-Aldrich F3165 Mouse AB_259529 Myc MBL M192-3 Mouse AB_11160947 Ub CST 3933S Rabbit AB_2180538 His CST 12698 Rabbit Gst CST 2624S Mouse AB_10692101 GAPDH(14C10) CST 2118 Rabbit AB_561053 Open in a separate window Antibodies used for immunoblot analyses Cell culture and in vitro manipulation.

Techniques:

Journal: The Journal of Neuroscience

Article Title: The Ubiquitin E3 Ligase TRAF6 Exacerbates Ischemic Stroke by Ubiquitinating and Activating Rac1

doi: 10.1523/JNEUROSCI.1751-17.2017

Figure Lengend Snippet: Antibodies used for immunoblot analyses

Article Snippet: The protein bands were detected using a Bio-Rad imaging system and specific protein expression levels were normalized to GAPDH protein. table ft1 table-wrap mode="anchored" t5 Table 3. caption a7 Antibody Manufacturer Catalog # Sources of species RRID TRAF6 Abcam Ab40675 Rabbit AB_778573 Bax CST 2772 Rabbit AB_329921 Bcl-2(50E 3 ) CST 2870 Rabbit AB_2290370 Cleaved Caspase3(Asp175) CST 9661 Rabbit AB_2341188 Caspase-3 CST 9662 Rabbit AB_331439 p-IkBa(ser32/36) CST 9246L Mouse AB_2267145 IkBa(L35A5) CST 4814 Mouse AB_390781 P-Ikkβ(Y199) Abcam Ab59195 Rabbit AB_943810 IKKβ CST 8943S Rabbit AB_11024092 P-NF-kB p65(Ser536) CST 3033 Rabbit AB_331284 p65 CST 4764 Rabbit AB_823578 P47-phox(P366) Bioworld BS3261 Rabbit AB_1662897 NOXA2/P67 phox Abcam {"type":"entrez-nucleotide","attrs":{"text":"Ab109366","term_id":"37572651","term_text":"AB109366"}} Ab109366 Rabbit AB_10860640 SD1(SOD1) Abcam Ab13498 Rabbit AB_300402 Heme Oxygenase 1(HO-1) Abcam Ab13243 Rabbit AB_299790 p-Rac1/cdc42(Ser71) CST 2461S Rabbit AB_2300703 Rac1/2/3 CST 2467 Rabbit AB_2176150 HA Sigma-Aldrich H6908 Rabbit AB_260070 Flag Sigma-Aldrich F3165 Mouse AB_259529 Myc MBL M192-3 Mouse AB_11160947 Ub CST 3933S Rabbit AB_2180538 His CST 12698 Rabbit Gst CST 2624S Mouse AB_10692101 GAPDH(14C10) CST 2118 Rabbit AB_561053 Open in a separate window Antibodies used for immunoblot analyses Cell culture and in vitro manipulation.

Techniques: Western Blot

Journal: Scientific Reports

Article Title: Basal autophagy prevents autoactivation or enhancement of inflammatory signals by targeting monomeric MyD88

doi: 10.1038/s41598-017-01246-w

Figure Lengend Snippet: Monomeric MyD88 is targeted by basal autophagy. ( a ) Monomerization of MyD88-GyrB promotes its degradation. Myd88 −/− MEFs stably expressing FLAG-tagged MyD88-GyrB were incubated with 10 μM novobiocin for the indicated periods, followed by cell lysis. Expression levels of FLAG-MyD88-GyrB and α-tubulin were assessed by immunoblotting. All the blots were obtained under the same experimental conditions, and the cropped images of the blots are shown. The uncropped images are in Supplementary Fig. . ( b ) Degradation of monomerized MyD88-GyrB is inhibited by lysosomal inhibition. Myd88 −/− MEFs stably expressing FLAG-tagged MyD88-GyrB were incubated with or without 10 μM novobiocin for 12 h in the presence or absence of 100 nM BafA1. Expression levels of FLAG-MyD88-GyrB and α-tubulin were assessed by immunoblotting. All the blots were obtained under the same experimental conditions, and the cropped images of the blots are shown. ( c ) Speckles of MyD88-GyrB are downregulated by monomerization. Myd88 −/− MEFs stably expressing FLAG-tagged MyD88-GyrB were incubated with 0.5% DMSO or 10 μM novobiocin for 12 h. Immunofluorescent staining of the cells for FLAG (green) and LAMP2 (red) was carried out, and cell nuclei were stained with Hoechst 33342. Images were obtained by confocal microscopy. Scale bar: 10 μm. ( d ) Monomerized MyD88-GyrB interacts with TRAF6. Myd88 −/− MEFs stably expressing FLAG-tagged MyD88-GyrB were incubated with or without 10 μM novobiocin for 12 h in the presence or absence of 100 nM BafA1, followed by cell lysis. Then immunoprecipitation (IP) using anti-FLAG-agarose was carried out with clarified lysates, followed by immunoblotting for TRAF6, p62/Sqstm1, LC3, ATG5, HSC70, LAMP2A, and FLAG-MyD88-GyrB. All the blots were obtained under the same experimental conditions, and the cropped images of the blots are shown.

Article Snippet: The membranes were blocked in EzBlockChemi buffer (ATTO) for 45 min. Immunoreactive bands were detected using the following primary antibodies and a horseradish peroxidase-conjugated secondary antibody: anti-MyD88 (D80F5) rabbit monoclonal antibody (mAb), anti-TIRAP (D6M9Z) rabbit mAb, anti-LC3A/B (D3U4C) rabbit mAb, anti-SQSTM1/p62 rabbit polyclonal Ab (pAb; 5114), anti-Atg5 (D5F5U) rabbit mAb, anti-HSPA8 (D12F2) rabbit mAb, and anti-α-tubulin (11H10) rabbit mAb obtained from Cell Signaling Technology; anti-TRAF6 rabbit polyclonal Ab (pAb) and anti-FLAG M2 mouse mAb obtained from SIGMA; anti-LAMP-2A rabbit pAb (51-2200) obtained from Thermo Fisher Scientific; anti-Actin (H-196) obtained from Santa Cruz Biotechnology.

Techniques: Stable Transfection, Expressing, Incubation, Lysis, Western Blot, Inhibition, Staining, Confocal Microscopy, Immunoprecipitation

Journal: Scientific Reports

Article Title: Basal autophagy prevents autoactivation or enhancement of inflammatory signals by targeting monomeric MyD88

doi: 10.1038/s41598-017-01246-w

Figure Lengend Snippet: TRAF6 facilitates resistance of MyD88 to degradation. ( a ) Basal MyD88 expression is lowered by TRAF6 deficiency. Expression of MyD88, TIRAP, TRAF6, and actin in Traf6 +/+ MEFs and Traf6 −/− MEFs was assessed by immunoblotting. All the blots were obtained under the same experimental conditions, and the cropped images of the blots are shown. The uncropped images are in Supplementary Fig. . ( b ) MyD88 speckles are downregulated by TRAF6 deficiency. Immunofluorescent staining of Traf6 +/+ MEFs and Traf6 −/− MEFs for MyD88 (green) and LAMP2 (red) was carried out. Cell nuclei were stained with Hoechst 33342. Images were captured by means of a confocal microscope. Scale bar: 10 μm. ( c ) Stably expressed MyD88-GyrB is downregulated by TRAF6 deficiency. Traf6 +/+ MEFs and Traf6 −/− MEFs were stably transfected with a plasmid encoding FLAG-MyD88-GyrB or with an empty plasmid. Expression of MyD88, TRAF6, and α-tubulin was assessed by immunoblotting. All the blots were obtained under the same experimental conditions, and the cropped images of the blots are shown. The uncropped images are in Supplementary Fig. . ( d ) Speckles of MyD88-GyrB are downregulated by TRAF6 deficiency. Traf6 +/+ MEFs and Traf6 −/− MEFs were stably transfected with the FLAG-MyD88-GyrB construct. Immunofluorescent staining for FLAG (green) and LAMP2 (red) was carried out, and cell nuclei were stained with Hoechst 33342. Images were acquired by means of a confocal microscope. Scale bar: 10 μm. ( e ) Basal MyD88 in TRAF6-deficient cells is restored by TRAF6. Traf6 −/− MEFs were transiently transfected with increasing amounts of a plasmid encoding FLAG-tagged wild-type TRAF6 (TRAF6 WT) or enzymatically inactive TRAF6 (TRAF6 C70A) or with an empty plasmid (mock). Expression of MyD88, FLAG-TRAF6, and α-tubulin was assessed by immunoblotting. Values within parentheses represent the ratio determined by densitometric measurement of the bands. All the blots were obtained under the same experimental conditions, and the cropped images of the blots are shown. The uncropped images are in Supplementary Fig. . ( e ) MyD88-GyrB expressed in TRAF6-deficient cells is restored by TRAF6. Traf6 −/− MEFs stably expressing FLAG-MyD88-GyrB were transiently transfected with a plasmid encoding TRAF6 WT or TRAF6 C70A, or an empty plasmid (mock). Expression of FLAG-MyD88-GyrB, endogenous MyD88, TRAF6, and α-tubulin was assessed by immunoblotting. All the blots were obtained under the same experimental conditions, and the cropped images of the blots are shown.

Article Snippet: The membranes were blocked in EzBlockChemi buffer (ATTO) for 45 min. Immunoreactive bands were detected using the following primary antibodies and a horseradish peroxidase-conjugated secondary antibody: anti-MyD88 (D80F5) rabbit monoclonal antibody (mAb), anti-TIRAP (D6M9Z) rabbit mAb, anti-LC3A/B (D3U4C) rabbit mAb, anti-SQSTM1/p62 rabbit polyclonal Ab (pAb; 5114), anti-Atg5 (D5F5U) rabbit mAb, anti-HSPA8 (D12F2) rabbit mAb, and anti-α-tubulin (11H10) rabbit mAb obtained from Cell Signaling Technology; anti-TRAF6 rabbit polyclonal Ab (pAb) and anti-FLAG M2 mouse mAb obtained from SIGMA; anti-LAMP-2A rabbit pAb (51-2200) obtained from Thermo Fisher Scientific; anti-Actin (H-196) obtained from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot, Staining, Microscopy, Stable Transfection, Transfection, Plasmid Preparation, Construct