FIGURE 8.

Matriptase is shed into the basolateral media while basolateral prostasin is transcytosed and shed into the apical media. Caco-2 cells were surface-biotinylated from the basolateral side at 4 °C. Cells were incubated at 37 °C after labeling and apical and basolateral media was collected at 5, 15, 30 min, 1, 2, 4, 8, and 18 h to reveal any shedding of the biotin-labeled matriptase, HAI-1 and prostasin. Biotinylated proteins from the media were pulled down with monomeric avidin agarose, separated with SDS-PAGE and analyzed by Western blotting. A, matriptase was released to the basolateral media within 1 h as three forms: a 70 kDa form and two proteolytically shed complexes at 85 and 110 kDa. B, two complexes of size 85 and 110 kDa could be detected with the M69 antibody within 1–2 h, suggesting these to be matriptase-HAI-1 complexes. C, no free 55 kDa HAI-1 was found in the media, only HAI-1 in the two complexes at 85 and 110 kDa was detected in the basolateral media after 1 h and accumulated up to 18 h. D, basolateral prostasin was detected in the apical media after 18 h incubation, confirming transcytosis before secretion into the apical media. Results shown are representative of two independent experiments.