Fig. 6. gp40 and MHC class I molecules do not co-precipitate. NIH 3T3 cells were infected for 5 h with wt-vac and the indicated recombinant vaccina viruses. As a control, NIH-3T3 cells stably expressing gp48 were used to show co-precipitation of MHC class I molecules with the complexed viral protein gp48. Cells were metabolically labeled for 3 h with [³⁵S]methionine/cysteine to cover both newly synthesized and matured proteins. Cells were lysed with digitonin and proteins were precipitated with the indicated antibodies (αgp40, peptide antiserum p11; αgp48, mAb CROMA229; αMHC, mAB 28-14-8S). Half of the proteins were digested with endo H and subsequently separated on 11.5–13.5% SDS–PAGE. Endo H sensitivity of MHC class I complexes is indicated as s, and resistance as r. The two differently glycosylated protein species of the m152 gene product (gp40/37) are marked as + and the slower migrating, highly glycosylated form as h. gp48 is identified as ^* and c indicates calnexin.