Deletion of the SAM domain inhibits TCR-mediated SLP-76 clustering. (A) J14 cells expressing SLP-76–GFP or ΔSAM–SLP-76–GFP fusion proteins were dropped onto coverslips coated with anti–human CD3ϵ antibody. Time point 0 represents the moment the cells made contact with the coverslip. GFP clustering at the cell/coverslip interface at indicated time points is shown. (B) Cells in panel A were activated on anti-CD3ϵ–coated coverslips for 10 minutes. Average intensity of the GFP clustering formed at the cell/coverslip interface was measured. The numbers of clusters within indicated intensity ranges were counted. Each line represents 1 analyzed cell. (C) Total thymocytes from SLP-76^+/+, SLP-76^m/+, and SLP-76^m/m littermates were either stimulated with anti-CD3ϵ cross-linking or left untreated. Cell lysates were subjected to immunoprecipitation with anti-SLP-76 polyclonal antibody, followed by Western blotting with anti–SLP-76 and anti-Gads antibodies. (D) Thymocytes and splenocytes from 4-week-old SLP-76^m/+, SLP-76^m/m, Gads^−/−, and SLP-76^m/mGads^−/− mice were analyzed by flow cytometry. (Top) Expression of CD4 versus CD8 on total thymocytes. (Middle) Expression of CD25 versus CD44 on CD4⁻CD8⁻ DN thymocytes. (Bottom) Expression of CD4 versus CD8 on splenocytes.