Immunologic characterization of P2X₁ receptor expression and processing in human platelets. (A) The predicted membrane topography of the P2X₁ receptor showing the extracellular glycosylation sites (◊) and the C-terminal tail location of the antigenic sites recognized by the anti-P2X₁antiserum. (B) Anti-P2X₁ receptor immunoblot of human platelet membranes (15 μg protein) versus rat vas deferens membranes (1 μg). (<<<, 95 kD; <<, 60 kD; < 45 kD). (C) Deglycosylation of P2X₁R protein expressed in platelets. Parallel aliquots of platelet membranes were directly processed for electrophoresis (right lane, −) or were denatured in 1% SDS and then incubated in the absence (left lane, −) or presence (middle lane, +) of endoglycosidase-F, as described under Materials and Methods. Twenty-five micrograms of protein was loaded in each lane. (<<<, 95 kD; <<, 60 kD; < 45 kD). (D) Comparative anti-P2X₁ receptor immunoblot of platelet membranes (30 μg) versus purified GPIIb/IIIa (400 ng) that were processed under reducing conditions. (<<<, 95 kD; <<, 60 kD). (E) Comparative anti-P2X₁ receptor immunoblot of platelet membranes (30 μg) versus purified GPIIb/IIIa (400 ng) that were processed under nonreducing conditions. (<<<, 95 kD; <<, 60 kD). (F) Comparative anti-CD61 immunoblot of the same platelet membrane (30 μg) and GPIIb/IIIa (400 ng) samples used in (E) (nonreducing conditions).