Fig. 3

Ser24-phosphorylated Dab2 is mainly distributed in the membrane fraction of platelets. a Cytosolic (Cyto) and membrane (Mem) proteins were isolated from the resting (R) and thrombin (Th, 1 U/ml)- (left panel) or PAR1 peptide (P1, 10 μM)- (right panel) stimulated human platelet lysates using the Mem-PER Plus Membrane Protein Extraction Kit protocol. The proteins were subject to Western blotting using the indicated antibodies. GAPDH and integrin αIIb was used as the marker for the cytosolic and membrane fractions, respectively. Representative data of 2–3 independent experiments are shown. b 293T cells were transfected with HA-Dab2 (wild-type) or HA-Dab2-S24A (S24A) expression plasmids and then stimulated with ethanol (E) or TPA (T, 1 μg/ml) for 30 min. Immunofluorescence staining was then performed using the mouse anti-Dab2 (red) and the rabbit anti-p-Dab2 (S24) (green) primary antibodies followed by the Alexa Fluor 546-conjugated anti-mouse IgG and the Alexa Fluor 488-conjugated anti-rabbit IgG secondary antibody. Nucleated cells were defined by positive fluorescent staining of Hoechst 33342 (blue). Arrows indicate the co-localization of Dab2 and p-Dab2 (S24). c The lysates (2 mg) from the resting (R) or thrombin-stimulated (Th, 1 U/ml) human platelets were immunoprecipitated by the control rabbit IgG (rIgG) or anti-p-Dab2 (S723) antibody. The immunoprecipitated proteins (IP) were analyzed by Western blotting using the indicated antibodies. Representative data of 3 independent experiments are shown