Defective TCR-mediated signaling in ΔSAM–SLP-76 T cells. (A) Purified SLP-76^m/+ and SLP-76^m/m CD4⁺ T cells were stimulated for 2 minutes by anti-CD3 cross-linking. Postnuclear lysates were analyzed by Western blotting with anti-pTyr, pSLP-76, pPLC-γ1, and pERK1/2 antibodies. SLP-76, PLC-γ, and ERK2 blots are shown as loading controls. (B-C) DP thymocytes and CD4⁺ splenic T cells from SLP-76^m/+ and SLP-76^m/m mice were loaded with Indo-1 and then stimulated by anti-CD3 cross-linking without (B) or with (C) the presence of 5 mM EGTA. Calcium concentration was monitored by flow cytometry and represented as the ratio of fluorescence at 405 nm and 510 nm. (D) Total thymocytes (left) and purified CD4⁺ T cells (right) from SLP-76^m/+ and SLP-76^m/m mice were stimulated for 2 minutes by anti-CD3 cross-linking. IP₃ was extracted, and IP₃ levels were measured.