Figure 10.

Galectin-4 is present on the extracellular side of mivrovillar vesicles. Mg²⁺-precipitated (Mg²⁺) and microvillar (Mic) membranes were resuspended in 25 mM HEPES and 150 mM NaCl, pH 7.0, containing 100 mM lactose and incubated for 10 min on ice. After centrifugation at 48,000 × g for 30 min, the pellets were collected, resuspended in the same buffer containing 100 mM lactose and 20 mM CHAPS, and incubated for 10 min at 37°C before centrifugation again as described above. The lactose-extracted fractions (lanes 1 and 4), lactose + CHAPS-extracted fractions (lanes 2 and 5), and the insoluble fractions (lanes 3 and 6) were analyzed by SDS-PAGE, followed by Western blotting using antibodies to aminopeptidase N (top) or galectin-4 (bottom). (The 140-kDa band is the transient high-mannose glycosylated form of aminopeptidase N, which is only present in the Mg²-precipitated fraction.) Molecular mass values are indicated.