Normal lymphocyte development in 1-month-old p66Shc^−/− mice. (A, left) Immunoblot analysis with anti-Shc antibodies of spleen, lymph nodes, and thymus postnuclear supernatants from untreated age-matched control (+/+) or p66Shc^−/− (−/−) mice. (Right) Immunoblot with anti-Shc antibodies of postnuclear supernatants from splenocytes from wild-type mice purified by Immunomagnetic sorting with anti-panB, anti-panT or negatively sorted using anti-panB plus anti-panT antibody-conjugated magnetic beads, as well as from splenic monocytes purified by adherence. Control blots of the same filters with antiactin mAb are shown below. Representative experiments are shown (n ≥ 3). (B) Flow cytometric analysis of thymocytes from 6-week-old control (+/+) or p66Shc^−/− (−/−) mice stained with anti-CD4/anti-CD8 and anti-CD3 fluorochrome-conjugated mAb. On the left, the histograms show the percentage (± SD) of double-negative (DN), double-positive (DP), or single-positive thymocytes for CD4 and CD8 expression. On the right the histogram shows the levels of CD3 expression: not expressed (CD3−) and with low (CD3L) or high (CD3H) expression. For each sample, fluorescence was analyzed on gated cells with forward and side light scatter properties of lymphocytes (n = 5). (C) Cells from spleen and lymph nodes of 6-week-old control (+/+) or p66Shc^−/− (−/−) mice were stained with anti-CD3/anti-CD22 and anti-CD4/anti-CD8 fluorochrome-conjugated mAb and analyzed by flow cytometry. The histograms show the percentage (± SD) of single-positive cells in spleen (left) and lymph nodes (right) analyzed on gated lymphocyte populations (n = 5; *P < .05; **P < .01).