<p><i>A</i>, Schematic of the approach used to target the I-A<sup>b</sup>-β locus. The exon structure of the I-A<sup>b</sup>-β locus is shown with the locations of the signal peptide (SP), extracellular domain (Extracellular), transmembrane domain (TM), and cytoplasmic tail (Cytoplasmic) indicated. The targeting construct consisted of a long arm of homology (LAH) consisting of exons 2–4, the K225R mutation introduced into exon 4, exons 5 and 6 fused to each other and to EGFP, a floxed self-excisable neomycin resistance cassette and a short arm of homology (SAH) consisting of the 3′UTR. Not drawn to scale. <i>B</i>, Splenocytes from I-A-β-EGFP or I-A-β-K225R-EGFP mice were stained with antibodies to detect B cell (B220), dendritic cell (CD11c) and T cell (CD3) populations and were analyzed by flow cytometry. Class II MHC, as detected from EGFP fluorescence, was present in B cells and dendritic cells, and class II MHC levels were increased in cells isolated from I-A-β-K225R-EGFP mice. <i>C</i>, Total splenocytes were isolated from heterozygous I-A-β-EGFP/HA-Ub and I-A-β-K225R-EGFP/HA-Ub mice and subjected to immunoprecipitation with an antibody against the MHC II α chain and subsequently to immunoprecipitation with an antibody against GFP. Total lysates were subjected to SDS-PAGE and immunoblot analysis with anti-GFP and anti-HA. All experiments were performed at least twice; representative experiments are shown.</p
