F-actin immobilization selectively inhibits PLCγ1 phosphorylation. (A and C) GFP-actin Jurkat T cells were either pretreated with 25 µM Y-27632 (Y-27) or left untreated and plated on OKT3-coated coverglasses. After 5 min, cells were treated with DMSO or JAS, then fixed at the indicated times, and stained for phospho-Y783 of PLCγ1 (A) or phospho-Y319 of Zap70 (C). Intensity of antibody staining is pseudocolored. Cont, control. Bars, 5 µm. (B and D) Analysis of fluorescence intensities from phospho-PLCγ1 (A) or phospho-Zap70 (C) staining. The IS outlines were gauged by F-actin intensity; total phosphoprotein intensity per IS was calculated for a mean of 45 cells (B) or 35 cells (D) per condition. Mean ± SEM is shown. *, P < 0.05; ***, P < 0.001.