Fig. 3

Biochemical characterization of ADAM10 in human platelets. (A) Analysis of ADAM10 N-glycosylation in control platelets: 100 µg of total platelet proteins were incubated with (+) and without (−) N-glycosidase F, loaded on 8% acrylamide gels and ADAM10 identified by immunoblotting. N-glycosidase F treatment causes a downward bandshift of 6 kDa of the ADAM10 immunoreactive band. Molecular weight markers are reported on the right. (B) Analysis of ADAM10 O-glycosylation in control platelets: 100 µg of total platelet were incubated with (+) or without (−) neuraminidase. 50 µg were removed and further incubated with (+) or without (− O-glycanase. Twenty micrograms of total proteins were loaded on 8% acrylamide gels and ADAM10 identified by immunoblotting. O-deglycosylation experiments failed to produce a shift of ADAM10 immunoreactive band. Molecular weight markers are reported on the right. (C) Western blot analysis performed with an antibody specific for ADAM10 in platelet subcellular fractions. Immunoreactivity is present in platelet homogenate (H) and in particulate (P), but not in the soluble fraction (S).