FIGURE 5:

WT A1AT and prosaposin are transiently reglucosylated by UGT1, whereas reglucosylation of NHK is more persistent. (A) MI8-5 Chinese hamster ovary cells were radiolabeled for 30 min. Where indicated, 0.5 mM DNJ was added 15 min before the end of the pulse. DNJ was also added at the indicated times of the chase, followed by 15-min incubation. Prosaposin was isolated with prosaposin antisera, followed by treatment with EndoH where indicated. Monoglucosylated prosaposin was isolated by GST-CRT pull down, followed by immunoprecipitation with prosaposin antisera. All samples were resolved on a 9% SDS–PAGE reducing gel. (B) The quantification of the percentage of reglucosylation of prosaposin was performed as in Figure 1, accounting for differences in amounts of sample used for each treatment as a percentage of EndoH-sensitive prosaposin, since this represents ER-localized protein compared with EndoH-resistant protein localized to the lysosome. The error bars are representative of the SD of three or more independent experiments. (C) MI8-5 Chinese hamster ovary cells where transfected with either WT or NHK A1AT and treated as in A. (D) The quantification of the percentage of reglucosylation of all bands was performed as in Figure 1 accounting for differences in amounts of sample used for each treatment. The error bars are representative of the SD of three or more independent experiments.