FIGURE 6.

Active prostasin is present on the basolateral as well as on the apical plasma membrane. Caco-2 cells on Transwell filters were surface biotinylated from either the apical or basolateral side at 4 °C. Biotinylated proteins were precipitated with monomeric avidin and gently eluted with biotin. The avidin pull-downs were divided into three: No further treatment (lanes 1 and 2), pull-down with aprotinin-Sepharose (lanes 3 and 4) and pull-down with trypsin inhibitor as negative control (lanes 5 and 6). The pull-down fractions were analyzed by SDS-PAGE and Western blotting. Prostasin was purified from both apical plasma membrane (lane 1) and basolateral plasma membrane (lane 2). The majority of the apical prostasin could be pulled down with the serine protease inhibitor aprotinin (lane 3). A small but significant fraction of basolateral prostasin was also able to bind aprotinin and hence was in its active form (lane 4). No prostasin binding was observed when using the negative control trypsin-coupled Sepharose (lanes 5 and 6). Results shown are representative of three independent experiments.