Figure 2.

Analysis of cell surface and intracellular PIR molecules. Viable splenocytes from normal adult BALB/c mice were radiolabeled with ¹²⁵I and solubilized in 0.5% NP-40. The cleared membrane lysates were incubated in wells precoated with 6C1 anti-PIR or an isotype-matched control mAb. The bound materials were resolved on SDS-PAGE using 10% acrylamide under nonreducing (not shown) and reducing conditions (A) or digested with or without N-glycanase before SDS-PAGE analysis (B). The same ∼85 and ∼125-kD PIR molecules were observed in splenocytes, purified splenic B cells, and granulocytes (C) as well as the X16C8.5 B cell and WEHI3 macrophage cell lines (D). In contrast, the 85-kD band was identified in PIR-A transfected fibroblasts, while the ∼120-kD band was expressed by the PIR-B transfectants (D). In the experiments shown in C and D, NP-40 lysates of splenocytes, purified B cells, granulocytes, the LTK cells transfected with empty vector or PIR-A1 or PIR-B expression vectors, the X16C8.5 B cell line, and the WEHI3 macrophage cell line were incubated in wells precoated with 6C1 anti-PIR mAb or isotype-matched control mAb (not shown). The bound PIR molecules were separated in SDS-PAGE under nonreducing conditions, transferred onto membranes, and identified with rabbit anti-PIR antiserum and enzyme-labeled goat anti–rabbit Ig antibody before visualization by enhanced chemiluminescence.