Fig. 2

PKC isoforms are responsible for the phosphorylation of Dab2-Ser24. a-c Human washed platelets were pretreated with staurosporine (100 nM) for 5 min and then stimulated with the indicated concentrations of the agonists. Platelet aggregation was recorded by using a platelet aggregometer (Chrono-Log) (panel a). After 10 min, human washed platelets were lysed and the platelet lysates were collected for Western blotting using the anti-Dab2 (p96) and anti-p-Dab2 (S24) antibodies. The proteolytic cleavage products of Dab2 were marked as *. The expression of β-actin was used as a control of equal protein loading. R, resting platelets (panel b). The level of Dab2-Ser24 phosphorylation was quantified by ImageJ software and normalized by the expression of β-actin. The level of Dab2-Ser24 phosphorylation in agonist-stimulated platelet lysates was arbitrarily set as 1. The data are presented as the mean ± SEM of 4 independent experiments. ***p < 0.001 (panel c). d GST-Dab2N (1 ~ 234 amino acids) recombinant protein was used as the substrate for the in vitro protein kinase assay using the indicated PKC isoforms. The level of Dab2-Ser24 phosphorylation was analyzed by Western blotting using the anti-p-Dab2 (S24) antibody. The expression of GST-Dab2N recombinant protein was used for the control of equal protein loading. e HA-Dab2 (S) protein or HA-Dab2-S24A (A) protein from the lysates of 293T cells transfected with HA-Dab2 or HA-Dab2-S24A expression plasmids was enriched by immunoprecipitation using the anti-HA antibody. The immunoprecipitated proteins were used as the substrates for the in vitro protein kinase assay using the indicated PKC isoforms. The level of Dab2-Ser24 phosphorylation was determined by Western blotting using the anti-p-Dab2 (S24) antibody. The expression of HA-Dab2 or HA-Dab2-S24A was used for the control of equal protein loading