Figure 4.

Binding of JSB1 and C219 in pancreatic β cells. (A) Distribution of JSB1 binding in a pancreatic β cell revealed by confocal immunocytochemistry. Note the punctuate distribution. (×2,000.) (B) Western blot analyses of β cell granular membranes from mouse islets with the mdr1-specific monoclonal antibodies JSB1 and C219. Islet granule membrane proteins (20 μg) were probed with 5 μg/ml JSB1 or 0.5 μg/ml C219. Arrows indicate the position of the 65-kDa granular membrane protein cross-reacting with the anti-mdr1 antibodies. (C) Photoaffinity labeling of plasma membrane (PM) and secretory granules (Gr) from mouse islets with [³H]glibenclamide. The positions of the molecular mass markers (MW), the stacking gel, and the tracking dye are indicated. In both experiments, a peak of specifically bound radioactivity was detected at ≈140 kDa. Because of the scarcity of material, the experiments were conducted only once (C) or twice (B). However, identical results were obtained when membranes from Ins1 insulinoma cells were used (n = 2, not shown).