Time course of Syk phosphorylation after CVX treatment. (A) Dami cells were stably transfected with equivalent levels of rGPVIa or rGPVIb isoforms (see supplemental Table 2). Cells were incubated for 30, 90, or 270 seconds in buffer containing 10 μg/mL CVX, and then lysed. Soluble proteins were isolated, and Syk was immunoprecipitated with specific antibody and electrophoresed on acrylamide gels, then transferred to PVDF membranes. The presence of tyrosine-phosphorylated Syk was determined by Western blot (WB) using monoclonal anti-phosphotyrosine 4G10 (top panel). The position of Syk is indicated by the horizontal arrow to the right of the gel. The membranes were then stripped and reblotted with monoclonal antibody specific for Syk (bottom panel). These data are taken from one representative example of 3 independent experiments. (B) Cumulative data analysis from 3 independent experiments. The density of 4G10-probed bands was determined by optical image analysis, as described in the text. The mean band densities in arbitrary units (ordinate) derived from lysates harvested at 30, 90, and 270 seconds after addition of CVX (abscissa) are plotted, in which ● represent data obtained from Dami cell lines stably transfected with rGPVIa, and ○ represent data obtained from cells transfected with rGPVIb. The vertical lines represent one standard deviation from the mean. The difference in mean band densities at 30 seconds and 90 seconds (∗) is statistically significant (P < .05).