Figure 5.

EDEM1 MLD promotes glycan trimming. Top and bottom left panels, empty plasmid (Mock), Man1b1-FLAG, EDEM1-FLAG, EDEM1(3K)-FLAG, MLD-FLAG, MLD(3K)-FLAG, and MLD(Cys-less)-FLAG were co-expressed with NHK in Man1a1/a2/b1/c1 KO CHO cells. 150 mm kifunensine (KIF) was added for 5 h prior to the pulse where indicated. Proteins were radiolabeled for 30 min with [³⁵S]Cys/Met and chased for 0, 1, and 2 h. Cells were lysed in MNT buffer, and lysates were divided equally and subjected to anti-FLAG and anti-A1AT immunoprecipitation. Proteins were resolved by reducing 8% SDS-PAGE. Bottom right panels, the NHK bands in the designated lanes were magnified, and a red line was drawn through the center of each 0-h time point and through the 4-h time point to illustrate mobility shifts. Gels are representative of three independent experiments.