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. 2016 Jun 22;291(34):17639–17650. doi: 10.1074/jbc.M116.720284

FIGURE 6.

FIGURE 6.

Lipid binding activity of Lck-SH2 domain is crucial for T cell signaling activities of Lck. A, calcium mobilization; C, Tyr phosphorylation of TCR signaling proteins in JCaM1.6 cells stably transduced with EGFP-tagged Lck WT and mutants after TCR stimulation. For calcium flux assay, cells were labeled with 4 μg/ml Indo-1 AM, and the calcium flux was monitored for 8 min after OKT3 (1 μg/ml) treatment by flow cytometry. For Tyr(P) monitoring, cells were stimulated with OKT3 (10 μg/ml for 5 min), and cell lysates were immunoblotted with indicated antibodies. B, calcium mobilization in JCaM1.6 cells stably transduced with EGFP-tagged Lck WT and A160K, respectively, after TCR stimulation with 0.1 μg/ml OKT3. D, JCaM1.6 cells stably transduced with EGFP-tagged Lck WT and A160K were stimulated with indicated doses of OKT3 for 5 min, and the levels of pERK1/2 were measured by intracellular staining and flow cytometry. To compensate for the differences in expression levels of WT and GOF mutants, cells expressing similar levels of EGFP-tagged proteins were gated and used for quantitative analysis. All data are from triplicate measurements that show essentially the same results. D, data represent means ± S.D. ***, p < 0.001 from Student's t test.