Figure 5.

Copurification of galectin-4 and brush border enzymes. A mucosal explant, labeled for 2 h with 0.5 mCi/ml [³⁵S]methionine, was homogenized in a Potter-Elvehjem homogenizer in 1 ml of 25 mM HEPES and 150 mM NaCl, pH 7.0, containing 10 μg/ml aprotinin and 10 μg/ml leupeptin, and centrifuged at 500 × g for 5 min. The supernatant was centrifuged for 48,000 × g for 30 min. The resulting pellet of total membranes was resuspended in 0.25 ml of HEPES buffer and solubilized for 10 min on ice by the addition of CHAPS (20 mM). The extract was centrifuged at 48,000 × g for 30 min to obtain a supernant of low temperature detergent-soluble protein and a pellet. The pellet was resuspended in 0.25 ml HEPES buffer and solubilized for 10 min at 37°C by 20 mM CHAPS and 1 mM EDTA. The extract was centrifuged at 48,000 × g for 30 min to obtain a supernatant of (solubilized) low-temperature detergent-insoluble protein and a pellet. Galectin-4 antibody (0.2 ml) was added to both the extracts of low-temperature detergent-soluble and low-temperature detergent-insoluble proteins, and after incubation at 4°C overnight, the extracts were centrifuged at 5000 × g for 5 min to pellet the immunoprecipitates. The immunoprecipitates of both the low-temperature detergent-soluble (lane 2) and low-temperature detergent-insoluble (lane 4) fractions were washed in buffer once and analyzed by SDS-PAGE together with 100 μl of the corresponding extract supernatants left after immunoprecipitation (lanes 1 and 3, respectively) and the pellet of detergent-insoluble material (lane 5). After electrophoresis and electrotransfer onto an Immobilon membrane, the gel tracks were visualized by autoradiography, and then Western blotted in three successive steps using the indicated primary antibodies. (The single sharp 36-kDa band seen in lanes 1 and 3 of the galectin-4 blot is residual staining of annexin II from the previous round of blotting because they were not observed in single blot experiments using the galectin-4 antibody alone). Molecular mass values are indicated.