<p><i>A</i>, Splenocytes from B6 (+), I-A-β-EGFP (WT) and I-A-β-K225R-EGFP (KR) mice were analyzed by flow cytometry for class II levels. Total levels of class II MHC were determined from GFP fluorescence and the levels of cell surface class II MHC was assessed by staining with antibodies against I-A-β. Total and cell surface levels of class II MHC are increased in both B cells and dendritic cells of I-A-β-K225R-EGFP mice relative to I-A-β-EGFP and B6. This analysis was performed 3 times with a total of 8 mice of each genotype; representative data are shown. <i>B</i>, Splenocytes were lysed in NP40 and analyzed by SDS-PAGE. Samples were either boiled, or incubated at room temperature (non-boiled) to distinguish between the class II MHC complex and free I-Aα and I-Aβ chains. GFP-tagged I-Aβ was detected with antibodies against GFP, while the untagged I-Aβ was detected with the JV2 antibody. Levels of the mutant I-Aβ increased in I-A-β-K225R-EGFP cells while wild type I-A-β levels in the same cells remained constant. <i>C</i>, Cells were analyzed as in <i>B</i>, and immunoblotted with antibodies directed against I-Aα. Levels of I-Aα increase in I-A-β-K225R-EGFP cells showing that both components of the class II MHC molecule are affected. Immunoblotting analysis was performed at least twice; representative data are shown.</p
