Down-regulation of MHC II synthesis during DC maturation. (A) Splenic DCs were metabolically labeled for 60 minutes immediately after purification or after culture for 2 to 6 hours. The cells were lysed and their MHC II molecules immunoprecipitated with mAb N22 and run in 12.5% SDS-PAGE. The amount of immunoprecipitate loaded was normalized for the efficiency of incorporation of radioactivity in each sample, measured by TCA precipitation of the cell lysates. The positions occupied by the α and β subunits of MHC II and the p31 (Ii) and p41 (Iip41) spliced variants of the MHC II chaperone Ii are indicated.¹  Note that the MHC II β-chain and Ii overlap in 12.5% SDS-PAGE.³⁴  The numbers at the bottom indicate the relative intensity of the region containing α, β, and Ii in each lane as determined in a phosphorimager. (B) The CD4^–CD8^–, CD4⁺, and CD8⁺ splenic DC subtypes were purified by preparative FACS and analyzed as in panel A immediately after purification or after overnight culture. (C) CD4⁺ and CD8⁺ DCs were purified from the spleens of PBS- or LPS-injected mice and metabolically labeled for 45 minutes. The MHC II molecules were immunoprecipitated with mAb N22 and run in 11% SDS-PAGE.