Phospho tyk2

Manufactured by Cell Signaling Technology

Sourced in United States

Phospho-TYK2 is a lab equipment product that detects the phosphorylation of the TYK2 protein. TYK2 is a member of the Janus kinase (JAK) family of tyrosine kinases and plays a role in cytokine signaling. This product can be used to measure the activation state of TYK2 in various experimental settings.

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Lab products found in correlation

Close spelling variants of this product

Phospho‐ Y1054/1055‐TYK2 (2 citations) Anti-phospho Tyk2 (2 citations)

7 protocols using phospho tyk2

Characterizing IL23R Signaling Pathways

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Proteins were detected by flow cytometry with Alexa Fluor 488 or phycoerythrin-labelled antibodies to IL23R (FAB41001P, R&D Systems), phospho-JAK1, phospho-JAK2, phospho-JAK3, phospho-TYK2, phospho-STAT1, phospho-STAT2, phospho-STAT3, phospho-STAT5, phospho-STAT6, FLAG (Cell Signaling) or phospho-STAT4 (BD Biosciences).
IL23R was immunoprecipitated using anti-IL23R antibody (Santa Cruz Technology, Santa Cruz, CA) bound to Protein A or Protein G Sepharose (EMD Millipore, Billerica, MA). Associated proteins were blotted with antibodies to JAK2, STAT3 (Cell Signaling), TYK2 (Abcam, Cambridge, MA), IL12Rβ1 (EMD Millipore), JAK3 (Santa Cruz Biotechnology) or IL23A (Proteintech). Control proteins were examined with glyceraldehyde 3-phosphate dehydrogenase or IL23R antibodies (EMD Millipore) as per ref 41 (link).

Sun R., Hedl M, & Abraham C. (2019). IL23 induces IL23R recycling and amplifies innate receptor-induced signalling and cytokines in human macrophages, and the IBD-protective IL23R R381Q variant modulates these outcomes. Gut, 69(2), 264-273.

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Western Blot and qRT-PCR Analysis of Signaling Pathways

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The protein samples from stimulated or transfected cells were extracted by using the Laemmli Sample Buffer (Bio-Rad Laboratories, Inc., USA). The concentrations of protein samples were quantified by using the BCA™ Protein Assay Kit (Beyotime #P0012). Antibodies purchased from Cell Signaling Technology (Beverly, MA) included: phospho-STAT3 (Tyr705) (#9145), phospho-STAT3 (Ser727) (#49081), STAT3 (#4904), phospho-JAK2 (Tyr1007/1008) (#3771), JAK2 (#3230), phospho-JAK1 (Tyr1034/1035) (#74129), JAK1 (#3344), phospho-TYK2 (Tyr1054/1055) (#68790), TYK2 (#14193), phospho-STAT1 (Tyr701) (#7649), STAT1 (#14994), phospho-AKT (Ser473) (#4060), AKT(Pan) (#4821), phospho-NF-κB p65 (Ser536) (#3033), NF-κB p65 (#8242), cyclin D1 (#2978), cyclin A2 (#4656), cyclin B1 (#12231), Caspase-3 (#29629), Bcl-xL (#2762), and Survivin (#2803). PARP antibody and GAPDH antibody were purchased from Beyotime (#AP102) and Genscript (#A00192), respectively. WB analysis was conducted as previously described^{58 (link)}. Three independent experiments were performed using samples collected at different days. qRT-PCR was conducted as described previously^{59 (link)}. All the primers used were listed in Fig. S1b.

She S., Zhao Y., Kang B., Chen C., Chen X., Zhang X., Chen W., Dan S., Wang H., Wang Y.J, & Zhao J. (2020). Combined inhibition of JAK1/2 and DNMT1 by newly identified small-molecule compounds synergistically suppresses the survival and proliferation of cervical cancer cells. Cell Death & Disease, 11(9), 724.

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Intracellular Phospho-Protein Analysis

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Intracellular proteins were detected in permeabilized cells by flow cytometry with Alexa Fluor 647-, phycoerythrin- or Alexa Fluor 488-labeled antibodies to phospho-JAK1, phospho-JAK2, phospho-JAK3, phospho-TYK2, phospho-ERK, phospho-p38, phospho-JNK and phospho-IκBα (Cell Signaling Technology, Danvers, MA).

Hedl M., Proctor D.D, & Abraham C. (2016). JAK2 Disease-Risk Variants are Gain of Function and JAK Signaling Threshold Determines Innate Receptor-Induced Proinflammatory Cytokine Secretion in Macrophages. Journal of immunology (Baltimore, Md. : 1950), 197(9), 3695-3704.

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Phosphorylation Signaling Pathway Analysis

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30–60 μg of lysates were subjected to SDS-PAGE and transferred to PVDF membranes (Thermo Scientific). The membranes were then probed using the recommended antibody dilution in 5% BSA in TBST (Tris-Buffered Saline and Tween 20) or 5% non-fat milk in TBST for phospho-STAT1 (Tyr701), STAT1, phospho-STAT2 (Tyr690), STAT2, phospho-Tyk2 (Tyr1054/1055), Tyk2, phospho-Jak1 (Tyr1022/1023), Jak1 or β-actin (13E5) (Cell Signaling). Validation of antibodies is provided on the manufacturer’s website.

Jarret A., McFarland A.P., Horner S.M., Kell A., Schwerk J., Hong M., Badil S., Joslyn R.C., Baker D.P., Carrington M., Hagedorn C.H., Gale M J.r, & Savan R. (2016). Hepatitis-C-virus-induced microRNAs dampen interferon-mediated antiviral signaling. Nature medicine, 22(12), 1475-1481.

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Western Blot Analysis of Protein Signaling

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Total protein was extracted from HL‐1 cells or PBMCs using standard protocols in a RIPA buffer with 1 mmol/L PMSF. Equal amounts of protein from different experimental groups were separated by 8%‐12% SDS‐PAGE and transferred to PVDF membranes (Millipore, Eschborn, Germany), which were blocked for 1 hour with 5% non‐fat milk in TBST at room temperature, then incubated overnight at 4°C with primary antibodies against TWEAK (Abcam, Cambridge, UK), Fn14 (Abcam, Cambridge, UK), ANP (Millipore, Temecula, CA), Troponin T (Novus, Littleton, CO, USA), β‐actin (Millipore), phospho‐JAK1 (at Tyr1022/1023), JAK1, phospho‐JAK2 (at Tyr1007/1008), JAK2, phospho‐TYK2 (at Tyr1054/1055), TYK2, phospho‐STAT1 (at Tyr701), STAT1, phospho‐STAT3 (at Tyr705), and STAT3 (all Cell Signaling Technology, Danvers, MA), and then incubated with appropriate secondary HRP‐conjugated secondary antibody for 1 hour at room temperature. The protein signals were detected using a chemiluminescence kit (Millipore, MA, USA).

Hao L., Ren M., Rong B., Xie F., Lin M., Zhao Y., Yue X., Han W, & Zhong J. (2018). TWEAK/Fn14 mediates atrial‐derived HL‐1 myocytes hypertrophy via JAK2/STAT3 signalling pathway. Journal of Cellular and Molecular Medicine, 22(9), 4344-4353.

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Flow Cytometry of Signaling Proteins

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Cell surface or intracellular proteins (permeabilized cells) were detected by flow cytometry with fluorophore-labeled Abs to phospho-ERK, phospho-p38, phospho-JNK, phospho-IκBα, phospho-STAT1, phospho-STAT4, phospho-JAK1, phospho-JAK2, phospho-TYK2 (Cell Signaling Technology, Danvers, MA), STAT1, STAT4, IL-10RA, LC3β, and NOS2 (Santa Cruz Biotechnology, Santa Cruz, CA).

Hedl M., Sun R, & Abraham C. (2020). Disease Risk-Associated Genetic Variants in STAT1 and STAT4 Function in a Complementary Manner to Increase Pattern-Recognition Receptor–Induced Outcomes in Human Macrophages. Journal of immunology (Baltimore, Md. : 1950), 205(5), 1406-1418.

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Comprehensive Analysis of JAK-STAT Signaling

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Cells were lysed in RIPA buffer (Thermo Fisher 89900) supplemented with protease/phosphatase inhibitor cocktail (Cell Signaling #5872). Lysates were sonicated, centrifuged to remove insoluble complexes, then boiled with NuPage sample buffer (Thermo Fisher NP0007) containing 20 mM DTT. The samples were subjected to gel electrophoresis and semi-dry transfer, and the resulting immunoblots were blocked in 5% BSA, then incubated overnight with primary antibody followed by HRP-conjugated secondary antibodies. Primary antibodies against the following targets were used: GAPDH (Cell Signaling D16H11), STAT1 (Santa Cruz C-111), phospho-STAT1 (Cell Signaling 58D6), phospho-STAT2 (Cell Signaling D3P2P), phospho-STAT3 (Cell Signaling D3A7), phospho-STAT5 (Cell Signaling C11C5), phospho-STAT6 (Cell Signaling 9361), JAK1 (Santa Cruz B3), JAK2 (Cell Signaling D2E12), TYK2 (Cell Signaling D4I5T), phospho-JAK1 (Cell Signaling 3331), phospho-JAK2 (Cell Signaling 3771), and phospho-TYK2 (Cell Signaling 9321). For the analysis of JAK phosphorylation, lysates were first incubated overnight with antibodies against total JAK protein conjugated to Protein G Dynabeads (Thermo Fisher 10007D). Immunoblotting was then performed as above.

Gruber C.N., Calis J.J., Buta S., Evrony G., Martin J.C., Uhl S.A., Caron R., Jarchin L., Dunkin D., Phelps R., Webb B.D., Saland J.M., Merad M., Orange J.S., Mace E.M., Rosenberg B.R., Gelb B.D, & Bogunovic D. (2020). Complex Autoinflammatory Syndrome Unveils Fundamental Principles of JAK1 Kinase Transcriptional and Biochemical Function. Immunity, 53(3), 672-684.e11.

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