Figure 4

Western blot analysis and measurement of CD45 and CD45RA and CD45RO activities. (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 various periods of time, as indicated. Cell lysates were sized by SDS-PAGE under reducing conditions, electrotransfered to nitrocellulose membranes and proteins revealed using an anti-CD45 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) Results of T cells of young (empty columns) and elderly (filled columns) donors are shown. Protein bands were analyzed by semi-quantitative densitometry and are reported in arbitrary units. Left panel shows the CD45/β-actin expression (**, p < 0.01), and right panel shows CD45 activity, in optical density units, of T cell lysate immunoprecipitates determined using 4-nitrophenyl phosphate as a substrate. Results of T cells of young (filled columns) and elderly (empty columns) donors are shown. (C) The CD45RA/CD45RO ratio in T cells from young and elderly is shown (top panel). CD45RO activity (middle panel) and CD45RA activity (bottom panel) shown in optical density units in T cell lysate immunoprecipitates determined using 4-nitrophenyl phosphate as a substrate. Results of T cells of young (filled columns) and elderly (empty columns) donors are shown. The protein transferred to nitrocellulose membranes were stained with Ponceau to further verify that similar amounts of protein had been measured for each point of activity. 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.