FIGURE 2.
Western blot analysis of a subset of the Stealth domain missense mutants. A shows representative Western blots (anti-V5) of HEK293 cell lysates ∼48 h after transfection with WT or mutant phosphotransferase αβ subunits with a C-terminal V5 tag. The anti-GAPDH control shows that the expression levels of the mutants were similar. B, endoglycosidase Hf (H) and peptide N-glycosidase F (F) treatment of HEK293 cell lysates expressing WT or W81L αβ phosphotransferase. Endoglycosidase Hf cleaved high mannose-type N-linked glycans, whereas peptide N-glycosidase F cleaved both high mannose and complex-type units. The asterisk indicates the slower migrating β subunit of W81L, which contains both high mannose and complex-type N-linked glycans. Note that there is a small amount of β subunit that migrates similar to WT.