Journal: Molecular Medicine Reports

Article Title: Τhe genetics of juvenile idiopathic arthritis: Searching for new susceptibility loci

doi: 10.3892/mmr.2017.7733

Figure Lengend Snippet: 3D representation both in volume and backbone of the TYK2 native structure (PDB code 4GVJ) (in brown) and the mutant model (in blue-grey) containing the position of the P1104A polymorphism (green). Helical segment 1099–1113 is in red. 3D, three dimensional.

Article Snippet: C_31565763_10, C_32036787_10 and C__60866522_10 for rs10919563, rs4750316 and rs34536443, respectively).

Techniques: Mutagenesis

Journal: iScience

Article Title: Abi1 mediates airway smooth muscle cell proliferation and airway remodeling via Jak2/STAT3 signaling

doi: 10.1016/j.isci.2022.103833

Figure Lengend Snippet:

Article Snippet: phospho-Tyk2 ((Y1054/Y1055) , Cell Signaling , #68790S/1: RRID: AB_2799752.

Techniques: Virus, Luciferase, shRNA, Control, Recombinant, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Kinase Assay, Software, Confocal Microscopy, Microscopy

Journal: Journal of Biological Chemistry

Article Title: Tyrosine Phosphorylation of Protein Kinase D2 Mediates Ligand-inducible Elimination of the Type 1 Interferon Receptor

doi: 10.1074/jbc.m111.263608

Figure Lengend Snippet: FIGURE 1. TYK2 activity is not required for IFN-induced recruitment of PKD2 to IFNAR1. A, phosphorylation and levels of endogenous IFNAR1 immunopurified (IP) from 11.1-derived cells that harbor wild-type (WT-5) or catalytically inactive (KR-2) TYK2 cells treated with IFN as indicated. B, immu- noblot analysis of IFNAR1 immunoprecipitated from 293T cells treated with IFN for the indicated times. Left lane of the upper panel represents the whole celllysatefromuntreated293Tcells.C,immunoblotanalysisofIFNAR1immu- nopurified from WT-5 and KR-2 cells treated with IFN for the indicated times. Whole cell lysates (WCL) were used for detection of tyrosine phosphorylation of STAT1 and total STAT1.

Article Snippet: Commercially available antibodies against Tyr(P)-463 in PKD1/PKC (Abcam; ab59415), anti-panTyr(P) (4G10), phospho-STAT1, STAT1 (Cell Signaling), FLAG, GST, and -actin (Sigma), HA (12CA; Roche Applied Science), Ser(P)-710 of PKD2 (BIOSOURCE), PKD2 (Bethyl Laboratories), PKD1/2, TYK2, and the intracellular domain of hIFNAR1 (Santa Cruz) were purchased.

Techniques: Activity Assay, Phospho-proteomics, Derivative Assay, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: Tyrosine Phosphorylation of Protein Kinase D2 Mediates Ligand-inducible Elimination of the Type 1 Interferon Receptor

doi: 10.1074/jbc.m111.263608

Figure Lengend Snippet: FIGURE 2. IFN stimulates kinase activity of PKD2 and its interaction with TYK2. A, activity of GST-PKD2 expressed in cells harboring wild-type or kinase-dead TYK2, and left untreated or treated with IFN, was analyzed by in vitro Ser-535 phosphorylation of GST-IFNAR1 as assessed by immunoblotting using a Ser(P)-535-specific antibody. Levels of substrate and kinase were also analyzed by immunoblotting. B, interaction between endogenous TYK2 and PKD2 in HeLa cells treated as indicated was analyzed by co-immunoprecipitation (IP) and immunoblotting. WCL, whole cell lysate. IgG, isotype antibody control. C, localization of endogenous TYK2 in 2fTGH cells or isogenic 11.1 cells was detected using primary anti-TYK2 antibody (1o Ab, where indicated) and secondary antibodies conjugated with Alexa Fluor 488. Nuclei were counterstained using DAPI. D, localization of endogenous PKD2 in 2fTGH cells transduced with the indicated shRNA was detected using primary anti-PDK2 antibody (1o Ab, where indicated) and secondary antibodies conjugated with Alexa Fluor 594. Nuclei were counterstained using DAPI. E, co-localization of TYK2 and PKD2 in 2fTGH cells treated with IFN (2000 units/ml for 5 min) or not was determined by confocal microscopy analysis using indicated antibodies.

Article Snippet: Commercially available antibodies against Tyr(P)-463 in PKD1/PKC (Abcam; ab59415), anti-panTyr(P) (4G10), phospho-STAT1, STAT1 (Cell Signaling), FLAG, GST, and -actin (Sigma), HA (12CA; Roche Applied Science), Ser(P)-710 of PKD2 (BIOSOURCE), PKD2 (Bethyl Laboratories), PKD1/2, TYK2, and the intracellular domain of hIFNAR1 (Santa Cruz) were purchased.

Techniques: Activity Assay, In Vitro, Phospho-proteomics, Western Blot, Immunoprecipitation, Control, Transduction, shRNA, Confocal Microscopy

Journal: Journal of Biological Chemistry

Article Title: Tyrosine Phosphorylation of Protein Kinase D2 Mediates Ligand-inducible Elimination of the Type 1 Interferon Receptor

doi: 10.1074/jbc.m111.263608

Figure Lengend Snippet: FIGURE 3. TYK2 activity is required for IFN-induced tyrosine phospho- rylation of PKD2. A, phosphorylation and levels of endogenous PKD2 immu- nopurified (IP) from WT-5 or KR-2 cells treated with IFN as indicated were analyzed by the indicated antibodies. B, tyrosine phosphorylation and levels of endogenous PKD2 immunopurified from HeLa cells treated with IFN as indicated were analyzed by immunoblotting. C, HA-tagged TYK2 was expressed in and immunopurified from 293T cells that were IFN-treated (lanes1–3and5)ornot(lane4).TYK2proteinswerethenincubatedwithPKD2 purified from serum-starved and untreated 2fTGH cells (except in lane 2) in the presence of ATP (except in lane 2). The reaction was analyzed by immu- noblotting using an anti-phosphotyrosine antibody. The recombinant Src protein was used as positive control.

Article Snippet: Commercially available antibodies against Tyr(P)-463 in PKD1/PKC (Abcam; ab59415), anti-panTyr(P) (4G10), phospho-STAT1, STAT1 (Cell Signaling), FLAG, GST, and -actin (Sigma), HA (12CA; Roche Applied Science), Ser(P)-710 of PKD2 (BIOSOURCE), PKD2 (Bethyl Laboratories), PKD1/2, TYK2, and the intracellular domain of hIFNAR1 (Santa Cruz) were purchased.

Techniques: Activity Assay, Phospho-proteomics, Western Blot, Purification, Recombinant, Positive Control

Journal: Journal of Biological Chemistry

Article Title: Tyrosine Phosphorylation of Protein Kinase D2 Mediates Ligand-inducible Elimination of the Type 1 Interferon Receptor

doi: 10.1074/jbc.m111.263608

Figure Lengend Snippet: FIGURE 4. TYK2 is capable of phosphorylating PKD2 on Tyr-438. A, alignment of fragments of primary sequences of the plekstrin homology domains within human PDK1 (Prkd2) and PKD2 (Prkd2),; Tyr-463 and Tyr438 are underlined. B, in vitro tyrosine phosphorylation of purified from cells GST-PKD2 (wild-type or Y438F mutant, YF) by recombinant truncated GST-TYK2833–1187 kinase analyzed by immunoblotting (IB). C, phosphorylation and levels of GST-PKD2 (wild type orY438Fmutant)expressedin293TcellstreatedornotwithIFNasindicatedbeforeharvesting.GST-PKD2specieswerepurifiedfromthelysatesusingaffinity beads and analyzed by immunoblotting using the indicated antibodies. D, interaction of endogenous IFNAR1 with exogenous GST-tagged PKD2 (wild type or Y438Fmutant)expressedin293Tcellsdeterminedbyimmunoprecipitation(IP)followedbyimmunoblottingusingtheindicatedantibodies.E,specificTyr-438 phosphorylation of GST-tagged PKD2 species (wild type or Y438F mutant) purified from 293T cells treated or not with IFN for 5 min analyzed by immuno- blotting using the indicated antibodies. F, specific Tyr-438 phosphorylation of endogenous PKD2 immunopurified from HeLa cells treated with IFN for the indicated times analyzed by immunoblotting using the indicated antibodies.

Article Snippet: Commercially available antibodies against Tyr(P)-463 in PKD1/PKC (Abcam; ab59415), anti-panTyr(P) (4G10), phospho-STAT1, STAT1 (Cell Signaling), FLAG, GST, and -actin (Sigma), HA (12CA; Roche Applied Science), Ser(P)-710 of PKD2 (BIOSOURCE), PKD2 (Bethyl Laboratories), PKD1/2, TYK2, and the intracellular domain of hIFNAR1 (Santa Cruz) were purchased.

Techniques: In Vitro, Phospho-proteomics, Purification, Mutagenesis, Recombinant, Western Blot

Journal: Poultry science

Article Title: Characterization and functional analyses of novel chicken leukocyte immunoglobulin-like receptor subfamily B members 4 and 5.

doi: 10.3382/ps/pez442

Figure Lengend Snippet: Figure 5. LILRB4R, -B4S, -B5R, and -B5S glycoprotein regulated the JAK-STAT signaling pathway. (A) Western blotting results of JAK2/TYK2, STAT1/3, and SOCS1 after LILRB4–5 transfection of HD11 cell line. (B) Changes in mRNA levels of JAK2/TYK2, STAT1/3 and SOCS1 genes after LILRB4–5 transfection in HD11 cell line were detected by qRT-PCR. Data are presented as the mean ± SEM (n = 3) of 3 independent experiments: *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: The following reagents were from the indicated manufacturers: mouse anti-chicken MHC Class I-PE and mouse anti-chicken β2m-PE antibody (Southern Biotech, Birmingham, AL, USA); rabbit antichicken STAT1 (phospho-Ser727), anti-chicken STAT3 (phospho-Ser727), and anti-chicken JAK2 (phosphoTyr1007/Tyr1008) antibody (Santa Cruz Biotech Dallas, Texas, USA); rabbit anti-chicken SOCS1, anti-chicken STAT1, anti-chicken STAT3 antibodies, horseradish peroxidase (HRP)-linked anti-rabbit secondary antibodies, and protein G–sepharose bead (Sigma-Aldrich, Louis, MO, USA); rabbit anti-chicken SHP2 (phosphoTyr542), anti-chicken JAK2, and anti-chicken TYK2 antibody (Biorbyt, San Francisco, CA, USA); rabbit anti-chicken GAPDH antibody (Abcam, Cambridge, MA, USA); Alexa Fluor® 488 Goat anti-rabbit IgG (H+L) secondary antibody (Invitrogen, Carlsbad, CA, USA); mouse monoclonal anti-chicken IFN-γ, IL-17A, IL-12p40 antibody and recombinants of these proteins (kindly provided by Dr. Hyun S. Lillehoj, USDA).

Techniques: Western Blot, Transfection, Quantitative RT-PCR