A cell-based reglucosylation assay. (A) For intact cells, HA was transiently transfected in both wild-type (WT) and MI8-5 (M) cells using the recombinant vaccinia virus–T7 RNA polymerase system and radiolabeled for 20 min in the presence of 0.5 mM DMJ at 34°C. The HA folding intermediates IT1, IT2, and NT and also the differences in glycosylation in wild-type and MI8-5 cells are indicated. (B) For SP cells, full-length HA mRNA was in vitro translated in the presence of wild-type or MI8-5 SP cells treated with 0.5 mM DMJ at 27°C. Radiolabeled HA was immunoprecipitated from lysates using a polyclonal antibody against HA. Untranslocated (UT) and native (NT) HA are marked. In A and B, black lines indicate that intervening lanes have been spliced out. (C) Full-length HA mRNA was translated in the presence of wild-type or MI8-5 SP cells for 60 min at 27°C. Samples were split, with half being chased for 5 h at 32°C. Samples were immunoprecipitated with HA antisera or the conformational antibodies F2 (IT2 and monomeric NT) and N2 (trimers). All samples were resolved via nonreducing (NR) and reducing (RD) 7.5% SDS-PAGE. (D) HA was translated in the presence of wild-type or MI8-5 SP cells in the presence or absence of DNJ and either immunoprecipitated with HA antisera or subjected to a GST-calreticulin pulldown. (E) HA was translated as in D. Radiolabeled HA treated with DNJ was alkylated with 20 mM NEM and immunoprecipitated with HA antisera. Radiolabeled HA produced in the absence of DNJ was subjected to an additional 3.5-h incubation with 1 mM cycloheximide to allow for complete glucose trimming. Samples were alkylated and subjected to immunoprecipitation by HA antisera. Samples were then split and treated with jack bean α-mannosidase (JBM) at 37°C for 18 h as indicated. All samples were resolved via 7.5% SDS-PAGE.