Western blot analysis of the levels of (active) tyrosine-phosphorylated (Y536) SHP-1. (A) Purified T cells of 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 an anti-Y536 SHP-1 mAb 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) Semi-quantitative densitometric analysis of protein bands of young (filled columns) and elderly (empty columns) donors reported in arbitrary units. Data are represented as the mean ± SD and are representative of one of 20 independent experiments. (C) Flow cytometry measurements of pSHP-1-Y536 MFI in T cells of young and elderly subjects at non-stimulated (NS) and stimulated for 30 seconds (S) states as decribed in the Materials and Methods section. The non-stimulated is normalized as 100% and significant difference is shown by **, p < 0.01. (D) Determination of SHP-1 activity in T cell lysate immunoprecipitate using 4-nitrophenyl phosphate as a substrate. Data show results of T cells of young (filled columns) and elderly (empty columns) donors reported in optical density units. Data are represented as the mean ± SD. Asterisks correspond to statistical significance (Student’s t-test) for p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***). Data are representative of one of 20 independent experiments.