(A) Purified CskAS (AS) or wildtype (WT) CD4+ T cells stimulated for 2 min with 1 μg/ml, 5 μg/ml or 10 μg/ml anti-CD3ε antibody in the presence of DMSO or 10 μM 3-IB-PP1 were analyzed by immunoblotting for the phosphorylation of the activation loop tyrosine of Lck and Fyn (Src pY416 antibody) and the inhibitory tyrosine of Lck (Lck pY505), phosphorylated ZAP-70 (ZAP70 pY319), LAT (LAT pY132) and PLC-γ1 (PLCγ1 pY783), as well as total actin (loading control). Data are representative of at least three independent experiments. (B) Purified total CskAS (AS) or wildtype (WT) T cells stimulated with the indicated dose of anti-CD3ε antibody for 2 min in the presence of DMSO or 5 μM, 1 μM or 0.4 μM 3-IB-PP1 were analyzed for phosphorylated ERK (p-ERK) by phosphoflow. Histograms were gated on CD4+ cells. Data are representative of at least three independent experiments for AS cells and two independent experiments for WT cells.