Expression of P2Y6 and P2X7 receptors in CD4+ T
Linsitinib and effect of antagonists on TCR-dependent intracellular Ca2+ mobilization. (A) Expression of P2Y6 and P2X7 receptors was analyzed by immunoblotting. Splenic CD4+ T cells were isolated by magnetic-activated cell sorting. Whole cell lysates of splenocytes (S) and splenic T cells (T) of BALB/c or C57BL/6 mice were separated on 10% SDS–PAGE and immunoblotted using anti-P2X7 receptor, anti-P2Y6 receptor or anti-HMGB1 (bottom panel) Ab. The result shown is typical of those obtained in three independent experiments. (B) Splenocytes loaded with Fluo-4 were stimulated with anti-CD3 and anti-CD28 mAbs, and then secondary Ab was added at 110 s after the first stimulation. The change in fluorescence was analyzed for an additional 3 min. The
cells were pre-incubated with apyrase (50 U/ml), MRS2578 (10 μM) or A438079 (100 μM) for 5 min. The data obtained from splenocytes of BALB/c mice are shown in the left panel, and those from splenocytes of C57BL/6 mice are shown in the right panel. Typical data from four independent experiments, all of which gave qualitatively similar results, are presented.
