Collagen-induced whole-cell, PLCγ2, Syk, and SLP-76 tyrosine phosphorylation in BTK^-/-, Tec^-/-, and Btk^-/-/Tec^-/- platelets compared with wild type and corresponding, densitometric analysis. Murine washed platelets from the 4 genotypes—wild-type, Btk^-/-, Tec^-/-, and Btk^-/-/Tec^-/-—were stimulated with either CRP (10 μg/mL) (A) or collagen (10 μg/mL) (B) or for 30 seconds and 90 seconds, respectively. All experiments were carried out in the presence of 10 μM lotrafiban to prevent aggregation. Basal and stimulated platelet samples were lysed by the addition of ice-cold lysis buffer and then used for the whole cell lysate (WCL) or the appropriate immunoprecipitation (IP). Tyrosine phosphorylation was revealed using the pan antiphosphotyrosine antibody 4G10. The presence of Btk and Tec was shown in the WCL using antibodies against these proteins. For the IP, a corresponding reprobe of PLCγ2, Syk, or SLP-76 was used to show equal loading. Results shown are representative of at least 3 identical experiments. Densitometry by band volume analysis was conducted on the tyrosine phosphorylation (4G10)-probed IP and the reprobes from panels Ai and Bi. Graphs show the ratio of the tyrosine phosphorylation band volumes to the corresponding reprobe band volumes then normalized to the wild-type control for each blot. Panel Aii shows the variation of phosphorylation of PLCγ2 (▪), SLP-76 (▦), and Syk (□) in the various knock-out genotypes after 30-second stimulation with CRP (10 μg/mL). Panel Bii shows equivalent data after stimulation with collagen (10 μg/mL). ^*Significant difference (P < .05) from control or between pairs. Each part shows the average band density from at least 3 Western blots ± SEM.