Western blot analysis of the levels of Y505- and Y394-phosphorylated forms of Lck in untreated and PTP-1-treated T cells. (A) T cells were left non-stimulated (NS) or exposed to a mixture of anti-CD3 and anti-CD28 (5 μg/ml each) mAbs for the times indicated, in the absence of the SHP-1 inhibitor (I) PTP-1 or its presence (50 ng/ml). Cell lysates were sized by SDS-PAGE under reducing conditions, electrotransfered to nitrocellulose membranes and proteins revealed using relevant mAbs and the chemiluminescence technique. β-Actin was used as control of gel loading. The protein transferred to nitrocellulose membranes were stained with Ponceau to verify that similar amounts of protein had been loaded in each lane. (B) Densitometric semi-quantification of the data, shown in arbitrary units in the case of young and elderly donors. For sake of clarity, significant statistical differences are indicated with filled circles (●, pLck-Y505) or filled squares (■, pLck-Y394) with respect to resting cells in the case of young donors. In the case of elderly donors, significant statistical differences are indicated with filled diamonds (♦, pLck-Y505) or ddags (‡, pLck-Y394) with respect to resting cells. (C and D) Densitometric ratio with respect to resting cells were determined for the various times and conditions of experiments for young (C) and elderly (D). Data are representative of one of 15 independent experiments.