Western blot analysis of the levels of tyrosine-phosphorylated (Y505) Lck. (A) Purified T cells from young and elderly donors were left non-stimulated (NS) or exposed to a mixture of anti-CD3 (5 μg/ml) and anti-CD28 (5 μg/ml) mAbs for 30 s or 5 min, as indicated. Cell lysates were sized by SDS-PAGE under reducing conditions, electrotransfered to nitrocellulose membranes and proteins revealed using appropriate 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) Time-related Y505-phosphorylated Lck analyzed by semi-quantitative densitometry and reported in arbitrary units in the case of stimulated (anti-CD3/anti-CD28, 5 μg/ml each) T cells of young (filled columns) and elderly (empty columns). Data are represented as the mean ± SD. Asterisks correspond to statistical significance (Student’s t-test) for p < 0.05 (*) and p < 0.01 (**). Data are representative of one of 20 independent experiments.