Figure 3.

Subcellular distribution of the type II Na/P_i cotransporter in untreated OK cells and in OK cells treated with PTH and/or leupeptin. OK cells were pretreated with nothing or leupeptin (100 μg/ml) for 30 min. After the pretreatment cells were incubated for 4 h with or without PTH (10⁻⁸ M) in the continued presence or absence of the lysosomal inhibitor. Before homogenization cells were surface-labeled with biotin. After centrifugation the Percoll density gradients were fractionated into three fractions of equal volume: fraction I containing low density membranes, fraction II containing membranes of an intermediate density, and fraction III containing high density membranes. Each of these fractions was analyzed for the activity of the lysosomal enzyme marker β-hexosaminidase, the presence of biotinylated cell-surface membranes, and the expression of the Na/P_i cotransporter. To determine the subcellular distribution of the transporter equal volumes of the gradient fractions were analyzed by SDS-PAGE and immunoblotting. In parallel, equal volumes of the same fractions were dot-blotted and probed with streptavidin–horseradish peroxidase to detect biotinylated cell-surface membranes. Depicted above are the distribution of the β-hexosaminidase, the Na/P_i cotransporter, and the biotinylated cell-surface membranes in untreated cells (A), in cells treated for 4 h with PTH (10⁻⁸ M) (B), in cells treated for 4.5 h with leupeptin (100 μg/ml) (C), and in cells pretreated for 30 min with leupeptin and treated thereafter for 4 h with PTH in the presence of the lysosomal inhibitor (D).