CD1d cytoplasmic lysines are required for MIR2-mediated downregulation and ubiquitination of CD1d. (A) Alignment and organization of the transmembrane and cytoplasmic tails of wild-type human CD1d and the lysine-to-arginine mutant. (B) Several chimeric proteins were created to confirm the requirement of cytoplasmic lysines for CD1d downregulation by MIR proteins. This schematic shows the theoretical domain organization of chimeric CD1d molecules that were constructed and the mutations that were introduced. (C) BJAB cells were cotransfected with either of the 2 chimeric CD1d molecules (wild type or lysine-to-arginine mutant) with an expression vector for EGFP (left panel) or MIR2 fused to EGFP (right panel). The levels of chimeric CD1d were determined by staining with anti-mouse CD1d antibodies, and the cells were analyzed by flow cytometry, gating upon EGFP-positive cells. (D) BJAB cells were stably transduced with either lacZ (as a control) or MIR2 and then stably transduced with either a wild-type chimeric CD1d or a lysine-to-arginine mutation. The cells were then lysed, chimeric mouse CD1d (mCD1d) was immunoprecipitated, and the immunoprecipitate was blotted for the presence of ubiquitin.