Localization of 1,4,5IP3R types in platelets.
(A) Western blot analysis of platelet 1,4,5IP3R isoform distribution in plasma (PM) and intracellular membranes (IM) prepared using high-voltage FFE. Numbers reflect molecular size markers in kilodaltons. Marker enzyme NADH cytochrome C reductase activity IM = 1.5 μmol/min per milligram protein; PM = 0.005 μmol/min per milligram protein. Actin was only detected in pm, not in IM. Ref. 33 shows full characterization of membranes. (B) Degradation of type III 1,4,5IP3R by endogenous proteases during membrane purification. PM were prepared using FFE, as outlined in “Materials and methods.” Samples loaded; lane 1, platelet lysate; lane 2, mixed membranes (MM) before FFE; lane 3, PM. Antibodies used in Western blot analysis reflect CT2 for type II and CT3 for the type III receptor. Numbers reflect positions of molecular size markers. (C) Intact platelets were surface labeled with biotin (see “Materials and methods”) and1,4,5IP3R were immunoprecipitated with R26, CT2, and R45 antibodies. Biotinylated proteins were detected using streptavidin–HRP.
