FIGURE 1.

Matriptase is located on the basolateral membrane whereas prostasin is apically located. Caco-2 cells grown on Transwell filters were biotin-labeled from either the apical (A) (lanes 1, 3, 5, 7, and 9) or basolateral (B) side (lanes 2, 4, 6, 8, and 10). Biotinylated proteins were precipitated with monomeric avidin, separated by SDS-PAGE and analyzed by Western blot with antibodies against total matriptase (M32) (lanes 1 and 2), matriptase-HAI-1 complex (M69) (lanes 3 and 4), the inhibitor HAI-1 (lanes 5–8) and prostasin (lanes 9 and 10) −/+ boiling of samples. Samples analyzed with the anti-prostasin antibody were boiled and reduced with DTT. The positions of molecular weight markers are indicated on the left. Protein and complex are marked with arrows and size on the right. The majority of the plasma membrane-bound matriptase was located on the basolateral membrane in a 70 kDa form, as detected with the antibody M32 (lane 2). A small fraction of the basolateral plasma membrane-bound matriptase was found in a 120 kDa form (lane 2). This form was also detected by the M69 (lane 4) and anti-HAI-1 (lane 6) antibodies. HAI-1 was found both on apical and basolateral plasma membrane (lanes 5–8). Apical HAI-1 (lane 5) migrated faster on the SDS-PAGE than the basolateral HAI-1 (lane 6) when samples were not boiled but displayed the same size after boiling of the samples (lanes 7 and 8). Plasma membrane-bound prostasin was located mainly on the apical side (lane 9), although minor amounts were detected on the basolateral membrane (lane 10). Results shown are representative of five independent experiments.