Analysis of Pyk2 (pY402) in human peripheral blood lymphocytes. Human peripheral blood mononuclear cells (PBMC) were either untreated (left panel) or stimulated by cross-linking of CD3 and CD28 with NA/LE Mouse anti-Human CD3 (Cat. No. 555329) and NA/LE Mouse anti-Human CD28 (Cat. No. 555725) on ice for 15 minutes followed by Purified Goat anti-Mouse Ig (Cat. No. 553998) on ice for 15 minutes, and then allowed to undergo phosphorylation at 37°C for 5 minutes (right panel). The cells were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes at 37°C, permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for 30 minutes, blocked with normal mouse immunoglobulin, and then stained with PE Mouse anti-Pyk2 (pY402, Cat. No. 560255) and APC Mouse anti-human CD3 (Cat. No. 340440). For data analysis, lymphocytes were selected by their scatter profile. The data demonstrates that the upregulated phosphorylation of Pyk2 was restricted to the CD3-positive T lymphocytes under these stimulation conditions. Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.
The specificity of mAb L68-1256.272 was confirmed by western blot analysis using unconjugated antibody on lysates from PBMC that were untreated (lane 1) or stimulated by cross-linking of CD3 and CD28 (lane 2). Pyk2 (pY402) is identified as a band of 116 kDa, with increased intensity in the stimulated cells.
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Analysis of Pyk2 (pY402) in human peripheral blood lymphocytes. Human peripheral blood mononuclear cells (PBMC) were either untreated (left panel) or stimulated by cross-linking of CD3 and CD28 with NA/LE Mouse anti-Human CD3 (Cat. No. 555329) and NA/LE Mouse anti-Human CD28 (Cat. No. 555725) on ice for 15 minutes followed by Purified Goat anti-Mouse Ig (Cat. No. 553998) on ice for 15 minutes, and then allowed to undergo phosphorylation at 37°C for 5 minutes (right panel). The cells were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes at 37°C, permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for 30 minutes, blocked with normal mouse immunoglobulin, and then stained with PE Mouse anti-Pyk2 (pY402, Cat. No. 560255) and APC Mouse anti-human CD3 (Cat. No. 340440). For data analysis, lymphocytes were selected by their scatter profile. The data demonstrates that the upregulated phosphorylation of Pyk2 was restricted to the CD3-positive T lymphocytes under these stimulation conditions. Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.
The specificity of mAb L68-1256.272 was confirmed by western blot analysis using unconjugated antibody on lysates from PBMC that were untreated (lane 1) or stimulated by cross-linking of CD3 and CD28 (lane 2). Pyk2 (pY402) is identified as a band of 116 kDa, with increased intensity in the stimulated cells.
准备和存储
推荐的实验流程
Either BD Cytofix™ fixation buffer or BD Phosflow™ Fix Buffer I may be used for cell fixation. Any of the three BD Phosflow™ permeabilization buffers may be used.
商品通知
- 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).
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
配套商品
Proline-rich tyrosine kinase 2 (Pyk2) is a member of the FAK subfamily of tyrosine protein kinases. Pyk2 responds to a wide variety of inflammatory and stress stimuli, such as cytokines, ligation of integrins or antigen receptors, mechanical strain, and cellular depolarization. A common characteristic of most Pyk2 stimuli is an increase in cytosolic free calcium. Pyk2 is involved in signaling pathways that regulate vital cellular functions in hematopoietic, epithelial, neuronal, endothelial, and other cell types. Activation of Pyk2 initiates its autophosphorylation at tyrosine 402 (Y402), followed by Src-mediated phosphorylation of other Pyk2 tyrosine sites, which enhances its activity and leads to downstream signal transduction through the MAPK and JNK pathways.
The L68-1256.272 monoclonal antibody recognizes the phosphorylated Y402 of activated Pyk2.
研发参考 (6)
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Anand AR, Cucchiarini M, Terwilliger EF, Ganju RK. The tyrosine kinase Pyk2 mediates lipopolysaccharide-induced IL-8 expression in human endothelial cells. J Immunol. 2008; 180:5636-5644. (Biology).
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Avraham H, Park S-Y, Schinkmann K, Avraham S.. RAFTK/Pyk2-mediated cellular signalling. Cell Signal. 2000; 12(3):123-133. (Biology).
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Faure C, Corvol J-C, Toutant M, et al. Calcineurin is essential for depolarization-induced nuclear translocation and tyrosine phosphorylation of PYK2 in neurons. J Cell Sci. 2007; 120:3034-3044. (Biology).
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Gluck SL. Acid sensing in renal epithelial cells. J Clin Invest. 2004; 114(12):1696-1699. (Biology).
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Orr AW, Murphy-Ullrich EM. Regulation of endothelial cell function by FAK and Pyk2. Front Biosci. 2004; 9:1254-1266. (Biology).
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Park, S-Y, Avraham HK, Avraham S. RAFTK/Pyk2 activation is mediated by trans-acting autophosphorylation in a Src-independent manner. J Biol Chem. 2004; 279(32):33315-33322. (Biology).
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