FIGURE 5.

Comparison of GnT-III modification among wild-type (WT), site-3 (D-3) and site-4 (D-4) deletion mutants. GnT-III was expressed in WT and D-3 and D-4 deletion mutants, and stable expression cells were established as described under “Experimental Procedures.” The expression levels of the GnT-III and GnT-III products in total cell lysates were detected with antibodies against GnT-III (A, upper panel) and α-tubulin to ensure equal loading (A, lower panel), respectively. B, the cell lysates were subjected to immunoprecipitation (IP) using an agarose-conjugated antibody against GFP. The immunoprecipitates were separated by 6.0% SDS-PAGE under non-reducing conditions. The membrane blot was probed with E4-PHA lectin and then was reprobed with an antibody against the α5 subunit. The ratio of E4-PHA to total α5 staining in WT cells was set equal to 1.0. C, the percentages of spread cells were quantified and expressed as the mean ± S.D. from three independent experiments. The bars represent S.D. The rounded cells were not considered as spread cells. The ratio of spread cells versus total cells (∼300 cells) of WT transfectants was set as 100.