Fig. 3.

Sequon skipping of adjacent NxS sites. (A) Diagram of ZAG showing the signal sequence (black), glycosylation sites, disulfide bonds (red lines) and Myc-DDK tag. The four sequons are numbered 1–4; ZAG mutants lacking one or more sequons are designated as ZAG-ΔXYZ, where XYZ is the list of mutated sequon(s). (B) HeLa cells were treated with negative control (NC) siRNA or siRNAs specific for STT3A or STT3B for 48 hours prior to transfection with wild-type ZAG. Cells expressing wild-type ZAG (B), or various ZAG glycosylation site mutants (C) were pulse labeled for 4 minutes and chased for 20 minutes. ZAG glycoforms (0–3 glycans) were immunoprecipitated using anti-DDK antibody and resolved by SDS-PAGE. Arrowheads designate a transient form of ZAG (see supplementary material Fig. S1). EH designates digestion with endoglycosidase H. Quantified values below gel lanes are for the displayed image that is representative of two or more experiments.