FIGURE 5.

CD300c and CD300b form cis-interacting complexes in the ER. A, COS-7 cells were transiently transfected with CD300 constructions. Forty-eight hours post-transfection cells were stimulated for 24 h with brefeldin A (1 μg/ml) or vehicle (70% EtOH). Cells were lysed (1% Triton X-100) and immunoprecipitated (IP) with the indicated antibodies. Western blots (WB) were conducted as described. Whole cell lysates (2%) were included as controls when assessing co-transfections. B, CD300c-HA and CD300b-FLAG were expressed both individually and simultaneously in COS-7. When expressed individually, clarificates where processed independently until the immunoprecipitation step with anti-HA.11 mAb. Western blots were conducted normally. C, immunoprecipitates from COS-7 co-transfected cells were subjected to peptide:N-glycosidase treatment in non-denaturing conditions. The eluted and immobilized fractions were analyzed separately (S = supernatant and B = beads) (left panel). COS-7 cells were transiently cotransfected with CD300 constructions. Eighteen hours post-transfection cells were stimulated for 48 h with tunicamycin (1 μg/ml) or vehicle (dimethyl sulfoxide). Cells were lysed and immunoprecipitated as described above (right panel).