Western blot analysis of the levels of tyrosine-phosphorylated PAG. (A) Purified T cells from young and elderly donors were left non-stimulated (NS) or exposed to a mixture of anti-CD3 and anti-CD28 (5 μg/ml each) 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) Protein bands were analyzed by semi-quantitative densitometry and are reported in arbitrary units. Results of T cells of young (filled columns) and elderly (empty columns) donors are shown. Data are represented as the mean ± SD. Asterisks correspond to statistical significance (Student’s t-test) for p < 0.05 (*), p < 0.01 (**). Data are representative of one of 20 independent experiments.