Figure 7.

Both wild-type and lectin-deficient Crt retard ER-to-Golgi transport of assembling class I molecules. (A) The indicated cell lines were radiolabeled for 10 min with [³⁵S]Met and chased with unlabeled Met for various times. Cells were then lysed and H-2K^b and D^b molecules were immunoisolated sequentially first with anti-8 antiserum followed by a combination of mAbs 28-14-8S and B22-249.R1. Isolated proteins were digested with endo H before analysis by SDS-PAGE. The mobilities of endo H-sensitive (s) and -resistant (r) heavy chains are indicated. Note that in Crt^−/− K42 cells two bands are designated as endo H resistant for D^b. These appear variably between experiments; the slower band corresponds to D^b molecules with all three oligosaccharides processed to complex forms whereas the faster band corresponds to molecules that possess 1 immature and 2 complex oligosaccharides. Both represent mature, Golgi-processed molecules and they are combined when calculating the percentage of endo H-resistant heavy chains. Asterisks denote nonspecific bands. (B) Fluorograms in A were scanned and endo H-sensitive and endo H-resistant band intensities were quantified using NIH ImageJ software (http://rsb.info.nih.gov/ij/). The endo H-resistant heavy chain was then calculated as a percentage of the total heavy chain signal at each time point. Error bars represent the SE values of three independent experiments for K^b and two independent experiments for D^b.