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Flow cytometric analysis of Stat5 (PY694) expression in human CD4+ T lymphocytes. Untreated (dashed line histogram) or treated (solid line histogram) peripheral blood mononuclear cells (PBMC) from the BD Phosflow™ T Cell Kit Lyophilized Cells (Catalog No. 560760) were reconstituted in BD Pharmingen™ Stain Buffer (Cat. No. 554656) for 15 minutes at room temperature. These cells are fixed, permeabilized, and lyophilized PBMC that are either untreated or treated with stimulators [including phorbol 12-myristate 13-acetate (PMA), interferon-alpha (IFN-α), interleukin-2 (IL-2), IL-4, and IL-6]. The reconstituted cells were then washed and stained with BD Horizon™ BUV395 Mouse Anti-Human CD4 antibody (Cat. No. 564724) and BD Horizon™ R718 Mouse Anti-Human Stat1 (pY701) antibody (Cat. No. 566977/566978). Histograms showing Stat5 (pY694) expression were derived from CD4-positive gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ Flow Cytometer System and FlowJo™ software.
BD Phosflow™ R718 Mouse Anti-Stat5 (pY694)
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- This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
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Stat (Signal transducer and activators of transcription) proteins mediate the biological activity of cytokines, including interleukins, interferons, erythropoietin, and growth factors. Ligand-receptor interaction activates constitutively-associated JAK family kinases and subsequent recruitment/activation of Stat proteins by tyrosine phosphorylation. Active Stat proteins then move to the nucleus to promote transcription of cytokine-inducible genes. Seven Stat proteins have been cloned, each of which is differentially expressed and/or activated in a cytokine-specific and cell type-specific manner. Stat5 has been characterized and shown to be encoded by two separate genes, Stat5a and Stat5b, which share over 90% identity at the amino acid level. Stat5a has been shown to be involved in lactogenesis and mammary development, while Stat5b has been shown to be involved in growth hormone signaling and liver gene expression. Both Stat5a and Stat5b are involved in IL-2 induced peripheral T cell proliferation. The peptide hormone, prolactin, binds to the prolactin receptor (PRLR) to initiate the lactogenic response. There are at least three forms of PRLR; however, only the long form activates the 92-kDa Stat5 protein by inducing phosphorylation at Y694. Once phosphorylated, Stat5 becomes an essential transcription factor which binds to the β-casein gene promoter. The presence of an SH2 domain within Stat5 suggests that it may directly interact with protein tyrosine kinases (PTKs) such as JAK2.
The 47 monoclonal antibody recognizes the phosphorylated Y694 of Stat5a. The homologous phosphorylation site in Stat5b is Y699.
The antibody was conjugated to BD Horizon Red 718, which has been developed exclusively for BD Biosciences as a better alternative to Alexa Fluor® 700. BD Horizon Red 718 can be excited by the red laser (628 – 640 nm) and, with an Em Max around 718 nm, it can be detected using a 730/45 nm filter. Due to similar excitation and emission properties, we do not recommend using R718 in combination with APC-R700 or Alexa Fluor® 700.
研发参考 (8)
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Bromberg J, Darnell JE. The role of STATs in transcriptional control and their impact on cellular function. Oncogene. 2000; 19(21):2468-2473. (Biology). 查看参考
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Gouilleux F, Wakao H, Mundt M, Groner B. Prolactin induces phosphorylation of Tyr694 of Stat5 (MGF), a prerequisite for DNA binding and induction of transcription. EMBO J. 1994; 13(18):4361-4369. (Biology). 查看参考
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Johnston RJ, Choi YS, Diamond JA, Yang JA, Crotty S. STAT5 is a potent negative regulator of TFH cell differentiation. J Exp Med. 2012; 209(2):243-250. (Clone-specific: Flow cytometry). 查看参考
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Liu KD, Gaffen SL, Goldsmith MA. JAK/STAT signaling by cytokine receptors. Curr Opin Immunol. 1998; 10(3):271-278. (Biology). 查看参考
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Prlic M, Bevan MJ. Exploring regulatory mechanisms of CD8+ T cell contraction. Proc Natl Acad Sci U S A. 2008; 105(43):16689-16694. (Clone-specific: Flow cytometry). 查看参考
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Suni MA, Maino VC. Flow cytometric analysis of cell signaling proteins. Methods Mol Biol. 2011; 717:155-169. (Clone-specific: Flow cytometry). 查看参考
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Van De Wiele CJ, Marino JH, Murray BW, Vo SS, Whetsell ME, Teague TK. Thymocytes between the -Selection and Positive Selection Checkpoints Are Nonresponsive to IL-7 as Assessed by STAT-5 Phosphorylation. J Immunol. 2004; 172(7):4235-4244. (Biology). 查看参考
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Wakao H, Gouilleux F, Groner B. Mammary gland factor (MGF) is a novel member of the cytokine regulated transcription factor gene family and confers the prolactin response. EMBO J. 1994; 13(9):2182-2191. (Biology). 查看参考
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