Fig. 4.

190-HARE(N2280A) cells coexpress a smaller variant with altered glycosylation that is delivered to the surface. A. Although HARE expression levels varied among the indicated stable cell lines, each expressed two membrane-bound 190-HARE(N2280A) bands that bind ¹²⁵I-HA in ligand blot (LB) assays. After exposure on film, western blot (WB) analysis with anti-V5 Ab visualized the HARE doublet. B. Both 190-HARE(N2280A) variants are on the cell surface. 190-HARE(N2280A) cells were incubated for 1 h at 4°C with HBSS containing 1 μg/mL mAb-30 with 0.055% digitonin (Weigel et al. 1983) [permeabilized to assess Total] or without [nonpermeabilized to assess Surface only]. Solubilized Ab-HARE complexes were captured using Protein A/G Sepharose. After centrifugation, the resin was eluted with 2× Laemmli buffer (Laemmli 1970), and the eluate was separated by SDS–PAGE, electro-blotted and HARE proteins detected by enhanced chemiluminescence using anti-V5 Ab. The ratios of the two HARE bands recovered in each lane were the same as assessed in digital images, and the Total signal was attenuated by ∼80% for comparison to the Surface signal (Harris et al. 2007; Harris et al. 2004). C. The smaller band has immature glycans. Immuno-purified nonreduced s190-HARE(N2280A) protein was subjected to SDS–PAGE after no treatment (lane 1) or digestion with PNGase-F (lane 2) or Endo-H (lane 3). The arrow indicates the minor smaller band, which was completely shifted by Endo-H, which did not affect the larger band