Effect of the removal of divalent cations on collagen- and CRP-stimulated tyrosine phosphorylation. Platelets were prepared and resuspended in Tyrode's-HEPES buffer without MgCl₂ in the presence of 1 mmol/L EDTA and 1 mmol/L EGTA. Where indicated, MgCl₂ (3 mmol/L) was added 5 minutes before stimulation. Tyrosine phosphorylation of (i) syk and (ii) PLCγ2 after stimulation by collagen (30 μg/mL) or CRP (3 μg/mL) for 90 seconds was measured using MoAb 4G10 as described in Fig 2. Reprobing for syk and PLCγ2 showed that similar levels of each protein were precipitated under all conditions. (The apparent reduction in tyrosine phosphorylation of syk by CRP in the presence of Mg²⁺ was due to a reduction of sample, as indicated by reprobing for syk [see also part (iii)]). (iii) The concentration response relationship for tyrosine phosphorylation of syk by CRP. Tyrosine phosphorylation was measured in syk immunoprecipitates using MoAb 4G10 as described in Fig 2. Results are representative of two to four similar experiments. A weakly tyrosine phosphorylated protein band that runs just above syk was detected in some but not all experiments (compare parts [i] and [iii]).