Fig. 1

Ser24 phosphorylation and proteolytic cleavage of Dab2 during agonist-induced human platelet activation. a The Dab2 Ser24 phospho-peptide (P-pep) but not the non-phospho-peptide (NP-pep) was recognized by a dot blot assay using the anti-p-Dab2 (S24) antibody. b 293T cells were transfected with HA-Dab2 (S24) or HA-Dab2-S24A (S24A) plasmids then stimulated with ethanol (E) or TPA (T, 1 μg/ml) for 30 min. Lysates of 293T cells were analyzed by Western blotting using the indicated antibodies. c, d Human washed platelets were treated with the indicated concentrations of TPA under the thermomixer assay condition. Lysates of human platelets were analyzed by Western blotting using the indicated antibodies (panel c) or the anti-p-Dab2 (S24) antibody which has been pre-incubated with H₂O (Control), P-pep or NP-pep as described in the peptide competition assays (panel d). e, f Human washed platelets were stimulated with the indicated concentrations of agonists and platelet aggregation was recorded by a platelet aggregometer (Chrono-Log). After 10 min, human washed platelets were lysed and the platelet lysates were collected for Western blotting using the indicated antibodies. The proteolytic cleavage products of Dab2 were marked as *. g, h Human washed platelets were pretreated with the indicated concentrations of MG-132 at RT for 30 min and then stimulated with TPA (25 ng/ml) and platelet aggregation was recorded by a platelet aggregometer (Chrono-Log). After 10 min, human washed platelets were lysed and the platelet lysates were collected for Western blotting using the indicated antibodies. Arrows indicate the addition of TPA (panel g). The proteolytic cleavage products of Dab2 were marked as * (panel h). i 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 the resting platelet lysate was arbitrarily set as 1. The data are presented as the mean ± SEM of 3–4 independent experiments. *p < 0.05; **p < 0.01