Functional reconstitution of early BCR signaling by full-length and s-SLP-65. (A) In vivo phosphorylation of SLP-65 proteins. SLP-65–deficient DT40 parental cells (slp-65^–/–, lanes 1-4) or transfectants stably expressing Flag-tagged versions of either full-length chicken SLP-65 (SLP-65, lanes 5-8) or the shortened form s-SLP-65 (lanes 9-12) (as in Figure 1) were left untreated (lanes 1, 5, and 9) or stimulated for the indicated times in minutes through their BCR (lanes 2-4, 6-8, and 10-12). Cleared cellular lysates were analyzed by immunoblotting with antibodies recognizing phosphotyrosine or the engineered peptide Flag in SLP-65 (top and bottom panels, respectively). (B) Association of SLP-65 isoforms with PLC-γ2 and Vav3. SLP-65–deficient DT40 cells (lanes 1-2 and 9-10) and the SLP-65–positive transfectants described in panel A as well as PLC-γ2– and Vav3-deficient DT40 cells (lanes 7-8 and 15-16, respectively) were left untreated or stimulated through their BCR for the indicated times. Cleared cellular lysates were subjected to immunoprecipitation with antibodies to PLC-γ2 (lanes 1-8) or Vav3 (lanes 9-16). SLP-65 proteins were detected by immunoblotting with antibodies to phosphotyrosine (top panels) or SLP-65 (middle panels). To control for immunoprecipitation efficiency and loading, PLC-γ2 and Vav3 were visualized by probing the membranes with anti–PLC-γ2 or anti-Vav3 antibodies (bottom panels). The relative molecular masses of marker proteins are indicated on the left. (C) Analysis of SLP-65–regulated Ca²⁺ mobilization. Cells described in panel A were stimulated through their BCR, and elevation of intracellular Ca²⁺ concentrations [Ca²⁺]_i were monitored by flow cytometry. Turquoise, blue, and red curves represent profiles of empty vector–transfected slp-65^–/– cells and transfectants expressing full-length or s-SLP-65, respectively.