Validation of the proteomics screen and characterization of DAPP1 as a PtdIns(3,4,5)P₃- and PtdIns(3,4)P₂-binding protein. (A) Human platelet lysates were incubated with control or PtdIns(3,4,5)P₃-coupled beads for 90 minutes at 4°C, with (+) or without preincubation with competing free PtdIns(3,4,5)P₃, before washing, elution, and western blotting analysis for a range of proteins identified in the proteomics screen. (B) Human platelet lysates were incubated with either PtdIns(3,4)P₂- or PtdIns(3,4,5)P₃-coupled beads as in panel A, and eluates were subjected to western blotting for DAPP1. (C) Human platelets were stimulated with 0.2 U/mL thrombin or 5µg/mL CRP for the indicated times, and lysates were blotted as indicated. The arrows indicate the molecular weight shift observed for DAPP1. (D) Human platelets stimulated with (i) thrombin (αT, 0.2 U/mL, 5 min) or (ii) CRP (5 μg/mL, 5 min) after 10 minutes of preincubation with dimethyl sulfoxide (DMSO) or 100 nM WTM were subjected to ultracentrifuge fractionation. Cytosol and membrane fractions were blotted as indicated. Histograms represent densitometry of blots from 3 independent experiments + standard error of the mean. Statistical analyses were performed by using 2-way analysis of variance with Bonferroni post-tests. *P < .05; **P < .001. Ctl, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.