Figure 1.

(A) Wild type and mutant tapasin expression levels were matched among paired transfectants expressing each type of MHC class I heavy chain (K^d or K^b). Aliquots of whole cell lysates were electrophoresed on a 10% acrylamide Tris-glycine gel. The proteins were subsequently transferred from the gel to a blot and probed with a mAb specific for mouse tapasin (Tsn). (B) More folded K^d was associated with tapasin C95S than with wild type tapasin. Immunoprecipitations were performed with monoclonal Ab 34-1-2 on lysates of the indicated cell types. The immunoprecipitated proteins were electrophoresed on a 10% acrylamide Tris-glycine gel, transferred to a membrane, and probed with anti-tapasin Ab (indicated as Tsn). (C) Cell surface K^b molecules on cells expressing mouse tapasin C95S had a slightly faster turnover rate, relative to cell surface K^b molecules on cells expressing wild type tapasin. MF cells that expressed mouse wild type tapasin or tapasin C95S were cultured in medium containing 2 μg/ml brefeldin A for 0, 3, 6, 9, 12, or 15 hours. Following incubation in the brefeldin A-containing medium, the cells were stained with anti-K^b mAb B8-24-3 and phycoerythrin-labeled secondary antibody, and analyzed on a FACS Calibur.