SLP-76 KI T cells show defects in TCR-induced proximal signal transduction and functional responses in vitro. (A) Contour plots show surface expression of CD4 and CD8 on lymphocytes from WT, Y145F, and Y112/128F spleens (n > 20). (B) Surface expression of TCRβ on CD4⁺ and CD8⁺ gated splenocytes from WT, Y145F, and Y112/128F mice (n = 6). (C) Spleen and lymph node cells from WT and KI mice were stimulated with anti-CD3 for the indicated times, lysed, and probed by Western blot with the indicated antibodies. Anti-Erk2 was used for a loading control (n = 3). (D) Ca²⁺ flux was measured in WT, Y145F, and Y112/128F CD4⁺ and CD8⁺ gated lymph node cells by flow cytometry following CD3, CD4, and CD8⁺ cross-linking (n = 4). (E) Splenocytes from WT, Y145F, and Y112/128F mice were incubated overnight in the presence of 0.1 μg of anti-CD3 or for 72 hours in the presence of 0.01 μg of anti-CD3 then measured for CD69 up-regulation (top panel; n > 5) and CFSE dilution (bottom panel; n > 5), respectively. Numbers in the tables represent frequency of cells within the gates depicted in the histograms.