Figure 8.

Modification of metabolically labeled ER proteins occurs only upon cell permeabilization. (A) HeLa cells expressing HA-tagged MHC class I heavy chains with lysine residues only in the luminal domain (HA-heavy chain; K-R) were labeled with [³⁵S]methionine and -cysteine. The cells were permeabilized with digitonin (Dig), as indicated, and treated with the amino group modifying reagent sulfo-NHS-biotin. The heavy chains were immunoprecipitated with HA antibodies. 20% of the immunoprecipitated material was analyzed directly by SDS-PAGE (lanes 1-4), and 80% was incubated with streptavidin beads to detect biotinylated material (lanes 5-8). Where indicated, one-half of each sample was treated with Endo H. All samples were subjected to SDS-PAGE followed by autoradiography. (B) Hybridoma B cells expressing IgG were analyzed as in A, except that the labeled IgG was collected with protein A/G-Sepharose.