antibodies against dok3  (Proteintech)

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: DOK3 is required for IFN-β production by enabling TRAF3/TBK1 complex formation and IRF3 activation.

doi: 10.4049/jimmunol.1301601

Figure Lengend Snippet: FIGURE 1. DOK3-deficient macrophages are impaired in IFN-b production and influenza virus clearance. (A) Semiquantitative RT-PCR analyses of dok3 mRNA in WT macrophages at 24 h postinfection with influenza virus. (B) Real-time RT-PCR analyses of ifn-b mRNA synthesis in WT and dok32/2 mac- rophages infected with influenza virus for various times. (C) ELISA measurement of IFN-b secretion by WT and dok32/2 macrophages after 24 h of influenza virus infection. (D) Semiquantitative RT-PCR analysis of influenza virus NS RNA in WT and dok32/2 macrophages at 12 h postinfection. Densitometry was performed on the band corresponding to the influenza NS gene and normalized to that of b-ACTIN and this was set as 1 for the respective uninfected samples. The numerical values after comparison with control samples are provided below each corresponding lane. (E) Quantitative real-time PCR analysis of influenza virus NS mRNA in WTand dok32/2 macrophages at 24 h postinfection. (F) Quantitative real-time PCR analyses of ifn-b mRNA synthesis in WT, dok32/2, and trif2/2 macrophages stimulated with naked poly(I:C) for 2 h. (G) ELISA measurement of IFN-b secretion by poly(I:C)-stimulated WT and dok32/2 mac- rophages at 6 h time point. Quantitative real-time PCR analyses of IFN-b mRNA synthesis in WT and dok32/2 macrophages stimulated with (H) liposome- enclosed poly(I:C), (I) poly(deoxyadenylic-thymidylic) acid [p(dA:dT)], and (J) CpG-ODN are shown. Control experiments represented uninfected or non- stimulated cells. Expression of ifn-b mRNA was normalized to that of b-actin mRNA. Triplicate experiments were performed. *p , 0.005.

Article Snippet: Abs against DOK3, ERK2, phosphoERK2, phospho-AKT (Thr308 and Ser473), TRAF3, JNK1, p38, IRF3, IkBa, and histone deacetylase 1 (HDAC1) were from Santa Cruz Biotechnology.

Techniques: Virus, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Infection, Enzyme-linked Immunosorbent Assay, Comparison, Control, Real-time Polymerase Chain Reaction, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: DOK3 is required for IFN-β production by enabling TRAF3/TBK1 complex formation and IRF3 activation.

doi: 10.4049/jimmunol.1301601

Figure Lengend Snippet: FIGURE 2. DOK3 is phosphorylated downstream of TRIF upon poly(I:C) stimulation. (A) WT macrophages were stimulated with naked poly(I:C) for various times and DOK3 was immunoprecipitated and examined for phos- phorylation via Western blot analysis using anti-phosphotyrosine (p-Tyr) Ab. The anti-DOK3 blot was included as control. (B) WT and trif2/2 macrophages were untreated or stimulated with naked poly(I:C) for 30 min and examined for DOK3 phosphorylation as in (A). (C and D) Semiquantitative real-time RT- PCR analyses of (C) Rantes and (D) ip10 mRNA in WT and dok32/2 mac- rophages at 2 h after stimulation with naked poly(I:C). (E) ELISA measure- ment of RANTES secretion by WT and dok32/2 macrophages stimulated with naked poly(I:C). (F) DOK3 phosphorylation in WT macrophages transfected with liposome-enclosed poly(I:C). Western blot analysis was performed as in (A). Data shown are representative of more than three independent experi- ments. *p , 0.005.

Article Snippet: Abs against DOK3, ERK2, phosphoERK2, phospho-AKT (Thr308 and Ser473), TRAF3, JNK1, p38, IRF3, IkBa, and histone deacetylase 1 (HDAC1) were from Santa Cruz Biotechnology.

Techniques: Immunoprecipitation, Western Blot, Control, Phospho-proteomics, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Transfection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: DOK3 is required for IFN-β production by enabling TRAF3/TBK1 complex formation and IRF3 activation.

doi: 10.4049/jimmunol.1301601

Figure Lengend Snippet: FIGURE 3. Defective IRF3 phosphorylation and nuclear localization in poly(I:C)-stimulated macro- phages lacking DOK3. (A) Confocal microscopy ex- amining IRF3 nuclear localization in nontreated (media) and poly(I:C)-stimulated WT, dok32/2, trif2/2, and tlr32/2 macrophages. Cells were stained with FITC-la- beled anti-IRF3 Ab (green). Nuclei of cells were stained with DAPI (blue). Original magnification 3100. (B) Western blot analysis examining nuclear IRF3 in poly(I:C)-stimulated WT and dok32/2 macrophages. Nuclear extracts from nontreated and stimulated cells were first probed with anti-IRF3 Ab and subsequently with anti-HDAC1 Ab to serve as loading control. (C) Western blot analysis examining IRF3 phosphoryla- tion in poly(I:C)-treated WT and dok32/2 macro- phages. Cells were stimulated for various times and whole-cell lysates were probed with an Ab that rec- ognized phospho-Ser396 residue of IRF3. The IRF3, tubulin, and b-actin blots were included as loading controls. Data shown are representative of more than three independent experiments. (D) IRF3 promoter/ DNA-binding activity was measured using poly(I:C)- stimulated nuclear extract proteins obtained at 2 h time point from WT and dok32/2 macrophages and assayed using an IRF3 TransAM (Active Motif) ELISA kit. Data shown are representative of two independent experiments.

Article Snippet: Abs against DOK3, ERK2, phosphoERK2, phospho-AKT (Thr308 and Ser473), TRAF3, JNK1, p38, IRF3, IkBa, and histone deacetylase 1 (HDAC1) were from Santa Cruz Biotechnology.

Techniques: Phospho-proteomics, Confocal Microscopy, Staining, Western Blot, Control, Residue, Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: DOK3 is required for IFN-β production by enabling TRAF3/TBK1 complex formation and IRF3 activation.

doi: 10.4049/jimmunol.1301601

Figure Lengend Snippet: FIGURE 4. DOK3 deficiency impairs the activation of TBK1 upstream of IRF3. Western blot analyses are shown of poly(I:C)-stimulated WT and dok32/2 macrophages examining (A) AKT activation using an Ab that recognized phospho-Thr308 (top panel) and phospho-Ser473 (bottom panel) residues of AKT and (B) TBK1 activation using an anti–phospho-TBK1 Ab. Blots were also probed with anti-AKT (A) and anti-TBK1 (B) Abs as loading controls. (C) DOK3 synergizes with TBK1 to induce ifn-b pro- moter activity. HEK293T cells were transfected with vector alone (control) or with vectors expressing DOK3 or TBK1 or both (with DOK3 in in- creasing dosage of 20, 50 and 100 ng) and together with plasmids bearing IFN-b promoter luciferase reporter (IFN-b-luc) and Renilla luciferase for normalization. Luciferase activity was measured at 24 h. Data shown are representative of more than three independent experiments. *p , 0.001.

Article Snippet: Abs against DOK3, ERK2, phosphoERK2, phospho-AKT (Thr308 and Ser473), TRAF3, JNK1, p38, IRF3, IkBa, and histone deacetylase 1 (HDAC1) were from Santa Cruz Biotechnology.

Techniques: Activation Assay, Western Blot, Activity Assay, Transfection, Plasmid Preparation, Control, Expressing, Luciferase

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: DOK3 is required for IFN-β production by enabling TRAF3/TBK1 complex formation and IRF3 activation.

doi: 10.4049/jimmunol.1301601

Figure Lengend Snippet: FIGURE 5. DOK3 is required for TRAF3/TBK1 complex formation and binds TRAF3 and TBK1 via its C-terminal domain. (A) Defective formation of TRAF3/TBK1 complex in poly(I:C)-stimulated dok32/2 macrophages. WT and dok32/2 macrophages were nontreated or stimulated with poly(I:C) for 60 min and TRAF3 was immunoprecipitated (IP) to examine TBK1 association (top panel) and vice versa with TBK1 immunoprecipitated to examine TRAF3 association (bottom panel) via Western blot analyses. (B) DOK3 binds TRAF3 and TBK1. HEK293T cells were cotransfected with FLAG-tagged DOK3 and HA-tagged TBK1 or HA-tagged TRAF3 and cell lysates were immunoprecipitated with anti-FLAG Ab and immunoblotted (IB) with anti-HA Ab to examine possible association of DOK3 with TBK1 and TRAF3. The FLAG immunoblot was included to show equivalent immunoprecipitation of FLAG- tagged DOK3 whereas whole-cell lysates (WCL) were immunoblotted to demonstrate expression of HA-tagged TBK1 or TRAF3 proteins. (C) Western blot analyses of endogenous TBK1/DOK3 interaction in primary macrophages that were nontreated (0 min) or stimulated for 60 min with naked poly(I:C). Western blots were performed as in (A). (D) Pictogram depicting FLAG-tagged full-length DOK3 protein or mutants harboring various combinations of PH, PTB, or tyrosine-rich carboxyl terminal domains. (E) The C-terminal domain of DOK3 acts in concert with TBK1 to further induce ifn-b promoter activity. HEK293T cells were transfected with empty vectors (control) or vectors carrying TBK1 and/or DOK3 or various mutant forms of DOK3 and assayed for the induction of luciferase activity as in Fig. 4C. Data shown are representative of three independent experiments. *p , 0.001. (F and G) The C-terminal domain of DOK3 binds TBK1 and TRAF3. HEK293T cells were transfected with either HA-tagged TBK1 (F) or HA-tagged TRAF3 (G) together with FLAG-tagged full-length DOK3 or various DOK3 mutants. Cell lysates were immunoprecipitated with anti-HA Ab and immunoblotted with anti-FLAG Abs to examine for association and with anti-HA for immunoprecipitation control. Whole-cell lysates were also included to ensure expression of the various transfected constructs. *NS, nonspecific band. Data shown are representative of three independent experiments.

Article Snippet: Abs against DOK3, ERK2, phosphoERK2, phospho-AKT (Thr308 and Ser473), TRAF3, JNK1, p38, IRF3, IkBa, and histone deacetylase 1 (HDAC1) were from Santa Cruz Biotechnology.

Techniques: Immunoprecipitation, Western Blot, Expressing, Activity Assay, Transfection, Control, Mutagenesis, Luciferase, Construct

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: DOK3 is required for IFN-β production by enabling TRAF3/TBK1 complex formation and IRF3 activation.

doi: 10.4049/jimmunol.1301601

Figure Lengend Snippet: FIGURE 6. BTK phosphorylates DOK3. (A) DOK3 is not phosphorylated in poly(I:C)-stimulated btk2/2 macrophages. Western blot analysis of DOK3 phosphorylation in WT and btk2/2 macrophages upon poly(I:C) stimulation is shown. Cells were stimulated with poly(I:C) and cell lysates were immuno- precipitated (IP) with an anti-DOK3 Ab or control IgG Abs and immuno- blotted (IB) with an anti-phosphotyrosine Ab (4G10). Membranes were subsequently stripped and immunoblotted with DOK3 Ab for loading control. (B) DOK3 is phosphorylated in poly(I:C)-stimulated but not BTK-inhibited macrophages. Cells were treated with DMSO as control or with the BTK- inhibitor LFM-A13, and DOK3 was immunoprecipitated and probed with anti-phosphotyrosine Ab and reprobed with anti-DOK3 Ab as control. (C) BTK kinase activity is required for DOK3 phosphorylation. HEK293T cells were nontransfected or transfected with FLAG-tagged DOK3 together with HA-tagged BTK, constitutively active BTK (E41K), or kinase-dead BTK (K430R). Cell lysates were immunoprecipitated with anti-FLAG Ab and immunoblotted with anti-phosphotyrosine (4G10) or anti-FLAG Abs. Whole- cell lysates (WCL) were included to show expression of the transfected gene constructs. (D) BTK physically binds DOK3. HEK293T cells were trans- fected with FLAG-tagged DOK3 and with or without HA-tagged BTK. Cell lysates were immunoprecipitated with anti-FLAG Ab and immunoblotted with anti-HA Ab. Whole-cell lysates were included to indicate expression of the transfected constructs. (E) In vitro kinase phosphorylation assay exam- ining the activities of purified WT and kinase-dead BTK (K430R) kinases on DOK3 as substrate as measured with the ADP-Glo kinase assay. Data are plotted as nanomoles phosphate transferred from ATP to a specific substrate. (F) BTK acts in concert with DOK3 and TBK1 to further increase ifn-b gene expression. HEK293T cells were transfected with various combinations of vectors bearing TBK1, DOK3, and BTK together with plasmids encoding IFN-b promoter luciferase (IFN-b-luc) reporter and Renilla luciferase and assayed at 24 h after transfection for luciferase activity. Data shown are representative of two independent experiments. * p , 0.05.

Article Snippet: Abs against DOK3, ERK2, phosphoERK2, phospho-AKT (Thr308 and Ser473), TRAF3, JNK1, p38, IRF3, IkBa, and histone deacetylase 1 (HDAC1) were from Santa Cruz Biotechnology.

Techniques: Western Blot, Phospho-proteomics, Control, Immunoprecipitation, Activity Assay, Transfection, Expressing, Construct, In Vitro, Kinase Assay, Gene Expression, Luciferase