Figure 1.

Characterization on GGL protein by SDS-PAGE and Western blotting. A: Structural analysis on GGL. Lane 1 shows the truncated recombinant GGL protein used for immunization (reduced). Samples in lanes 2 to 5 were run under reducing conditions and those in lanes 6 to 9 were run under nonreducing conditions. Lanes 2 and 6 were samples from WT spleen, lanes 3 and 7 from GGL^−/− spleen. Lanes 4 and 8 were samples from WT uterus, lanes 5 and 9 from GGL^−/− uterus. B: Deglycosylation of GGL with endoglycosidase H and N-glycosidase F. Samples in lanes 1 to 4 were WT spleen homogenates and those in lanes 5 to 8 were WT uterus homogenates. Lanes 1 and 5 show untreated samples. Lanes 2 and 6 show samples incubated with the buffer in the absence of enzymes. Lanes 3 and 7 show samples treated with endoglycosidase H, and lanes 4 and 8 show samples treated with N-glycosidase F. C: Dissociation of GGL from the cell membrane. The left panel shows the result of releasing GGL protein from a uterus membrane preparation with dithiothreitol. Lane 1 is untreated sample, lane 2 is the supernatant after dithiothreitol treatment, and lane 3 is the pellet after dithiothreitol treatment. The right panel shows the result of limited papain digestion on a uterus membrane preparation. Lane 1 is the untreated sample, lane 2 is the supernatant from a papain-digested sample, lane 3 is the pellet from a papain-digested sample, lane 4 is the supernatant of the sample incubated with buffer in the absence of papain, and lane 5 is the pellet. The numbers at the left side of the figures indicate the molecular weights in kd. Twenty μg of total protein from spleen homogenates and 10 μg of total protein from uterus homogenates were analyzed in all of the experiments.