Role of IDE in expression of “proteasome-independent” HLA class I alleles.
HLA-A3-expressing HEK293 cells and HLA-B27-transfected HeLa cells were transfected with 4×100 nM nM siRNA. Sixty-five hours later, 1 µM epoxomicin or 10 µg/ml brefeldin A were added to the cultures for 2 h or 30 min, respectively, before removal of most cell surface class I molecules by acid stripping and another 6 h incubation with the same drugs. HLA class I expression was detected using mAb W6/32 immediately after acid stripping (A, C left panel) and after the 6 h incubation (A, C center panel). The panels on the right show HLA class I re-expression relative to untreated cells as histograms. The panels in B and D show an evaluation of HEK293 cells using mAb GAP.A3 with specificity for HLA-A3 (B) and of HeLa-B27 cells using mAb ME-1 recognizing HLA-B27 (D). AS, acid stripping; BFA, brefeldin A; EPO, epoxomicin; filled histogram, secondary Ab only. One out of 3 experiments is shown.