Mouse/ABCC4 expression at the platelet plasma membrane. (A) Immunoblots of whole-platelet lysates, loaded at the indicated concentrations, derived from Abcc4 WT and KO mice probed with Abcc4, Abcg2, Abcc5, Abcc1, and actin antibodies. (B) After densitometry and actin normalization, the amount of each protein was quantified. The error bar represents one standard deviation from n = 3 independent samples. (C) Immunoblot of biotinylated membranes bound with streptavidin beads and intracellular contents (SN) prepared from mouse Abcc4 WT platelets and quantified and plotted as a bar graph. Each experiment was performed 3 times with PRP pooled from 3 to 5 mice for each genotype. (D) Relative amount of plasma membrane ABCC4, Na⁺/K⁺-ATPase, and P-selectin was determined by densitometry from multiple individual samples (n = 4) with the error bar indicated. (E) Super resolution structured illumination microscopy (SIM) showing localization of ABCC4 (green), Na⁺/K⁺-ATPase (blue), and actin (Red). Two cross-sectional views (XZ and YZ) show ABCC4 localized with Na⁺/K⁺-ATPase at the plasma membrane (gray arrows). The dashed white line demarcates the point of platelet coverslip attachment, the white line represents a tracing that outlines the platelet shape (see supplemental Figure 1A), and the yellow arrow represents the intracellular portion of the platelet identified by actin staining. The scale bar corresponds to 1 μm for all 3 views.