Figure 2.

(A) Western blotting for mouse tapasin showed that soluble tapasin was present at a level similar to (or slightly exceeding) wild type mouse tapasin in transfected MF cells, and Western blotting for the transfected mouse MHC class I heavy chain demonstrated that L^d, K^d, or K^b heavy chains were expressed at a roughly similar level in the MF cells. After electrophoresis of samples of cell lysates on a 10% acrylamide Tris-glycine gel for tapasin or on a 4→20% acrylamide Tris-glycine gel for MHC class I heavy chain, the proteins were blotted and probed with Ab specific for mouse tapasin (Tsn) or with 64-3-7 (for HC). The L^d heavy chain naturally has the 64-3-7 epitope, and the epitope was introduced into the K^d and K^b heavy chains by site-directed mutagenesis. (B) Murine soluble tapasin did not stabilize mouse TAP. Lysate samples were electrophoresed on a 10% acrylamide Tris-glycine gel before blotting and probing with antibody specific for mouse TAP. (C) Soluble mouse tapasin did not associate with TAP. Immunoprecipitates of TAP molecules were electrophoresed on a 10% acrylamide Tris-glycine gel. The proteins were then transferred to a blotting membrane and probed with antiserum specific for mouse tapasin (Tsn). (D) The folding of L^d, K^d, and K^b heavy chains was improved by the intracellular presence of wild type tapasin but not soluble tapasin. Immunoprecipitations were performed with mAb 30-5-7, 34-1-2, and Y3 (for folded L^d, K^d, and K^b, respectively) on lysates of MF cells that expressed each of the aforementioned MHC class I heavy chains along with no tapasin, wild type tapasin, or soluble tapasin. Immunoprecipitations with the 64-3-7 mAb were also done on corresponding cell lysates. The immunoprecipitates were electrophoresed on 4→20% acrylamide Tris-glycine gels, transferred to membranes, and probed with 64-3-7. (E) Tapasin association with the mouse MHC class I heavy chain was abrogated by the deletion of the tapasin TM/CYT region. Open MHC class I heavy chains were immunoprecipitated from a lysate of each of the indicated cell types with the 64-3-7 mAb. The immunoprecipitates were electrophoresed on a 10% acrylamide Tris-glycine gel, transferred to a membrane, and probed with an anti-tapasin antibody (Tsn).