Figure 3.

(A) Tapasin and K^d were expressed at similar levels in K^d+wt hTsn relative to K^d+sol hTsn. Samples of lysates from MF, MF+K^d, MF+K^d+wt hTsn, and MF+K^d+sol hTsn were electrophoresed on a 10% acrylamide Tris-glycine gel for tapasin (Tsn) or on a 4→20% acrylamide Tris-glycine gel for K^d, the proteins were transferred to a blotting membrane and probed with Ab specific for human tapasin (Tsn) or with 64-3-7 to identify the total epitope-tagged K^d heavy chain (HC). (B) Soluble human tapasin binds very weakly to K^d. Open K^d heavy chains were immunoprecipitated from a digitonin lysate of each of the indicated cell types with the 64-3-7 mAb in the presence of 200 μM DSP. The immunoprecipitates were electrophoresed on acrylamide Tris-glycine gels, transferred to membranes, and probed with 64-3-7 to identify the immunoprecipitated open K^d heavy chain (HC) or with anti-tapasin mAb (Tsn) to identify the co-immunoprecipitated tapasin. (C) Soluble human tapasin did not enable K^d to be expressed at the surface of mouse cells. Cells were incubated with secondary antibody only, or an antibody against the folded form of K^d (34-1-2), or an antibody against the open form of K^d (64-3-7). Results obtained with the secondary antibody only were all less than 5.0. Values on the y axis are relative mean fluorescence intensity (MFI) units. Wt hTsn = wild type human tapasin; Sol hTsn = soluble human tapasin. Very similar results were obtained in a separate flow cytometry experiment.