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• W1976236427 abstract "Biochemical studies have demonstrated that phosphorylation of lymphocyte cell kinase (p56lck) is crucial for activation of signaling cascades following T cell receptor (TCR) stimulation. However, whether phosphorylation/dephosphorylation of the activating or inhibitory tyrosine residues occurs upon activation is controversial. Recent advances in intracellular staining of phospho-epitopes and cytometric analysis, requiring few cells, have opened up novel avenues for the field of immunological signaling. Here, we assessed p56lck phosphorylation, using a multiparameter flow-cytometric based detection method following T cell stimulation. Fixation and permeabilization in conjunction with zenon labeling technology and/or fluorescently labeled antibodies against total p56lck or cognate phospho-tyrosine (pY) residues or surface receptors were used for detection purposes. Our observations showed that activation of Jurkat or primary human T cells using H2O2 or TCR-induced stimulation led to simultaneous phosphorylation of the activating tyrosine residue, Y394 and the inhibitory tyrosine residue, Y505 of p56lck. This was followed by downstream calcium flux and expression of T cell activation markers; CD69 and CD40 ligand (CD40L). However, the extent of measurable activation readouts depended on the optimal stimulatory conditions (temperature and/or stimuli combinations). Treatment of cells with a p56lck-specific inhibitor, PP2, abolished phosphorylation at either residue in a dose-dependent manner. Taken together, these observations show that TCR-induced stimulation of T cells led to simultaneous phosphorylation of p56lck residues. This implies that dephosphorylation of Y505 is not crucial for p56lck activity. Also, it is clear that cytometric analysis provides for a rapid, sensitive, and quantitative method to supplement biochemical studies on p56lck signaling pathways in T cells at single cell level. © 2012 International Society for Advancement of Cytometry" @default.
• W1976236427 created "2016-06-24" @default.
• W1976236427 creator A5012620834 @default.
• W1976236427 creator A5087670157 @default.
• W1976236427 creator A5091809007 @default.
• W1976236427 date "2012-06-06" @default.
• W1976236427 modified "2023-10-16" @default.
• W1976236427 title "TCR-induced T cell activation leads to simultaneous phosphorylation at Y505 and Y394 of p56lck residues" @default.
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• W1976236427 doi "https://doi.org/10.1002/cyto.a.22070" @default.
• W1976236427 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/22674786" @default.
• W1976236427 hasPublicationYear "2012" @default.
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