Stat4

Manufactured by Santa Cruz Biotechnology

Sourced in United States

STAT4 is a protein involved in the signal transduction and activation of transcription process. It acts as a transcription factor and plays a crucial role in the differentiation and function of T helper cells.

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5 protocols using stat4

Western Blot Analysis of STAT Signaling

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Preparation of whole cell lysates and performance of western blot analysis were as described in ref. ^{51 (link)}. Cell extracts (20–40 µg/lane) were fractionated on 4–12% gradient SDS-PAGE, and antibodies used were: pSTAT3 (Cell Signaling Technology, Danvers Massachusettes); IL-12p35, Ebi3, Cyclin E, cyclin -D1, P27Kip1, pSTAT1, pSTAT3, pSTAT4, STAT4, and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (Supplementary Table 2). Pre-immune serum was used in parallel as controls and signals were detected with HRP-conjugated secondary F(ab′)₂ Ab (Zymed Laboratories) using the ECL-PLUS system (Amersham, Arlington Heights, IL, USA).

Dambuza I.M., He C., Choi J.K., Yu C.R., Wang R., Mattapallil M.J., Wingfield P.T., Caspi R.R, & Egwuagu C.E. (2017). IL-12p35 induces expansion of IL-10 and IL-35-expressing regulatory B cells and ameliorates autoimmune disease. Nature Communications, 8, 719.

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STAT Activation Pathway Analysis

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Viable CD4 T cells were purified on a Ficoll gradient and treated with lysis buffer (RIPA buffer; 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% Sodium deoxycholate, 0.1% SDS) containing protease inhibitor mixture (Roche), 1 mM NaF (Sigma-Aldrich) and 1 mM NA₃VO₄ (Sigma-Aldrich). Protein samples were separated by SDS-PAGE and transferred to a PVDF membrane (Millipore). Primary antibody directed against STAT3, phospho-STAT3 (Cell signaling Technology), STAT4 (Santa Cruz) and phospho-STAT4 (Santa Cruz) and then HRP-cojugated Donkey anti-rabbit antibody (Affinity Bioreagents) were used to detect target protein by ECL detection kit (GE Healthcare).

Lee Y.K., Landuyt A.E., Lobionda S., Sittipo P., Zhao Q, & Maynard C.L. (2017). TCR-independent functions of Th17 cells mediated by the synergistic actions of cytokines of the IL-12 and IL-1 families. PLoS ONE, 12(10), e0186351.

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Immunoblotting of STAT Proteins

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Total cell lysates were prepared in lysis buffer (20 mM Hepes, pH 7.4, 20 mM NaCl, 10% Glycerol, and 1% Triton X-100) and protease and phosphatase inhibitor cocktails (CalBiochem). Blots were probed with antibodies that are specific for p-STAT1 (Y701, BD Biosciences), STAT1 (BD Biosciences), STAT2 (Santa Cruz), STAT3 (BD Biosciences), STAT4 (Santa Cruz), STAT5 (BD Biosciences), STAT6 (BD Biosciences), GAPDH (Santa Cruz), and β-actin (sigma).

Xiao W., Klement J.D., Lu C., Ibrahim M.L, & Liu K. (2018). IFNAR1 controls autocrine type I interferon regulation of PD-L1 expression in myeloid-derived suppressor cells. Journal of immunology (Baltimore, Md. : 1950), 201(1), 264-277.

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CD4+ Th1 Cell Signaling Profiling

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A western analysis was performed on primary CD4⁺ Th1 cells maintained in high IL-2, low IL-2, or low IL-2 with αKG for two days. The antibodies used were for HK2 (Abcam, ab76959), TCF1 (Cell Signaling, C63D9), CTCF (Millipore, 07–729) and STAT4 (Santa Cruz, C-20, sc-486X). Total STAT4 protein expression was monitored as a control for equivalent protein input in each sample in the western analysis. For flow cytometry, primary CD4⁺ Th1 cells maintained in high IL-2, low IL-2, or low IL-2 with αKG for two days were analyzed for CXCR3 (αCXCR3; Clone: CXCR3–173; Biolegend). Flow cytometry analyses were done with FlowJo V.10. software.

Chisolm D.A., Savic D., Moore A.J., Ballesteros-Tato A., León B., Crossman D.K., Murre C., Myers R.M, & Weinmann A.S. (2017). CCCTC-Binding Factor Translates Interleukin 2- and α-Ketoglutarate-Sensitive Metabolic Changes in T Cells into Context-Dependent Gene Programs. Immunity, 47(2), 251-267.e7.

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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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