ceacam1^−/− platelets display hyper-phosphorylated proteins including PLCγ2 after CRP stimulation over time. (A) Tyrosine phosphorylation in whole-cell platelet lysates from wild-type and ceacam1^−/− mice were stimulated with CRP (10 μg/mL) over 3 minutes. Then 30 μg of each platelet lysate was loaded onto a 10% sodium dodecyl sulfate–polyacrylamide gel and tyrosine phosphorylation was detected on the western blot, using a horseradish peroxidase (HRP)–conjugated anti-phosphotyrosine RC20 antibody. Erk-2 blot (bottom panel) was included as a protein loading control. This is a representative blot of 3 experiments. (B) PLCγ2 was immunoprecipitated from lysates of wild-type or ceacam1^−/− platelets stimulated by CRP (10 μg/mL) at time (T) 0 and 90 seconds and immunoblotted for phosphotyrosine using a HRP-conjugated anti-phosphotyrosine RC20 antibody. PLCγ2 antigen is included as a loading control of the immunoprecipitates (bottom panel). Relative intensity of tyrosine phosphorylated PLCγ2 to total PLCγ2 antigen was quantitated using ImageJ software version 1.40g (available from http://rsbweb.nih.gov/ij/) from 3 replicate experiments.