FIG. 2.

L^d/B7.2 chimeras are sensitive to mK3, and this correlates with their ability to associate with TAP/tapasin. (A) Schematic representation of L^d/B7.2 chimeras constructed with the luminal and TM regions of H-2L^d and cytoplasmic tail of either mouse B7.2 (L^d/mB7.2) or human B7.2 (L^d/huB7.2). A sequence alignment of the cytoplasmic tails of the indicated proteins is shown below the diagram. Lysine residues (K) are shown in bold. Identical or similar residues are shaded. (B) Constructs in panel A were introduced into B6/WT3 cells, followed by transduction with pMIG only (vector) or pMIG-mK3 (mK3). Sorting was used to enrich for GFP⁺ cells. Cells were incubated for 24 h with 100 U of IFN-γ/ml and then analyzed for expression of wt L^d or the L^d/B7.2 constructs by using MAb 30-5-7. Solid lines, staining of vector-only cells; dashed lines, staining of the mK3-expressing cells. (C) IFN-γ-treated cells were pulse-labeled with [³⁵S]Cys/Met and chased for the indicated times with unlabeled Cys/Met. L^d/huB7.2 molecules were precipitated with MAb 64-3-7. Precipitates were resolved by SDS-PAGE and visualized by autoradiography. L^d/huB7.2 bands are indicated (upper panel). In the lower panel, relative band intensities from the gels are plotted as a percentage of the intensity at time zero for each cell line. (D) Digitonin cell lysates from cells were immunoprecipitated with anti-L^d MAb 64-3-7. Precipitates were then blotted with the antibodies listed. Loading of each sample was normalized on the basis of an equal level of L^d/B7.2 molecules. (E) The L^d/huB7.2-TM construct was introduced into L cells. Surface L^d expression was monitored (MAb 30-5-7) after transduction of the cells with pMIG (vector) or pMIG-mK3. Right panels show anti-tapasin immunoblotting of anti-TAP-1 or anti-L^d precipitates from the indicated cell lines. The L^d/huB7.2-TM construct consists of the luminal domain of L^d (residues 1 to 282) followed by the TM and cytoplasmic tail of human B7.2 (residues 225 to 306).