Proximal TCR signaling through ZAP-70 and LAT is impaired by treatment with PF-562,271. A) Western Blot analysis of total phospho-tyrosine levels relative to tubulin levels in human peripheral blood T cells in the presence or absence of 10 μM test compounds. Blots are representative of three independent experiments. B) Western Blot analysis of ZAP-70 at Y319 and LAT phosphorylation at Y191 relative to total protein levels in human peripheral blood T cells. Blots are representative of three independent experiments. C) Western Blot analysis of ERK and D) p38 MAPK phosphorylation relative to total protein levels in human peripheral blood T cells. Blots are representative of three independent experiments. E) Quantification of ZAP-70, LAT, and ERK phosphorylation (mean +/− SD, n=3, p < 0.05 relative to DMSO control (*) or relative to TCR control (#), ANOVA).