Figure 3.

Differential ubiquitination of HLA-B7 and HLA-B27 heavy chains. (A) BM macrophages expressing HLA-B27 and HLA-B7 were treated for 21 hours with IFNγ, and then an additional 3 hours without or with bafilomycin (100 nM) (Baf) or bortezomib (10 nM) (Bor). HLA class I heavy chains immunoprecipitated with HC10 and analyzed under non-reducing or reducing conditions as indicated, using 3B10.7. (B) Samples from experiment described in (A) were immunoblotted with anti-ubiquitin antibody (α-Ub). (C) The ratio of ubiquitinated material (B) (from entire lane) to immunoprecipitated total heavy chain quantitated under non-reducing conditions (A, reduced) is plotted for HLA-B27 and HLA-B7 under each experimental condition. Results represent mean ± SEM (error bars) from five experiments. (*, p < 0.05) (D) Cells treated as described in (A) were used to immunoprecipitate all ubiquitinated material, and then blotted with HC10 to visualize heavy chains. In the long exposure image the red color indicates saturation.