Hyperglycosylation of GRP94. (A) The domain structure and location of glycosylation sites in GRP94. (B) The Δlog₂ values for GRP94 sites in STT3A^−/− and STT3B^−/− cells. Error bars designate SDs (n = 3–8) unless denoted by an asterisk (n = 2). Not quantifiable (NQ) and not detected (ND) report results for the STT3B^−/− cells. (C) Protein (50 µg) in lysates from WT and mutant cells was analyzed by protein immunoblotting using antisera to GRP94 and the α-subunit of ATP synthase. EH designates digestion with EH. (D) EH digestion time course of pulse-labeled GRP94. (E) Pulse labeling of GRP94 in similar numbers of WT and mutant cells. The right hand lane is a lighter version of the preceding lane. (F) Pulse labeling (5 min) of GRP94 in STT3A^−/− cells followed by the indicated chase times. (G) Pulse labeling of human GRP94-DDK-His and GRP94-DDK-His N217Q mutant in WT and mutant cells. (H) Pulse labeling of GRP94 in WT or STT3A^−/− cells treated with the following compounds: DTT (200 µm), tunicamycin (Tn, 0.6 µM), thapsigargin (Tg, 0.1 µM), and NGI-1 (10 µM). (I) Pulse labeling of GRP94 in control fibroblasts (C-1 and C-2), STT3A-CDG fibroblasts, and WT and STT3A^−/− cells. The EH-digested sample is from the STT3A^−/− cells and is equivalent to 10% of the undigested sample.