(A) The MHC class I negative cell line 721.221 and the tapasin and HLA-A and -B negative cell line 721.220 were transduced with HLA-A2 and –B7. shRNA specific for TAPBPR (shTAPBPR) was used to produce TAPBPR depleted versions of these cell lines. TAPBPR was isolated by immunoprecipitation from these four cell lines. Western blot analysis was performed for TAPBPR, tapasin, HLA-B (3B10.7), HLA-A2 (HCA2), and calnexin on lysates and TAPBPR immunoprecipitates as indicated. This data is representative of three independent repeats. Cytofluorometric analysis of (B) HLA-A2 (detected with BB7.2) or (C) HLA-B7 (detected with anti-Bw6 antibody) on .221 (A2+ , B7+ ) (grey filled histogram), .221 (A2+ ,B7+) shTAPBPR (red line histogram), .220 (A2+ ,B7+ ) (blue line histogram), .220 (A2+ ,B7+ ) shTAPBPR (green line histogram). Staining on the non-transduced 721.221 and 721.220 cells with BB7.2 and Bw6 (grey solid line) or with an isotype control (grey dashed line) are included as controls. The data is representative of three independent experiments. (D) Thermal stability of HLA-A2 expressed in .221 (A2+ ,B7+ ) and .220 (A2+ ,B7+) -/+ stable depletion of TAPBPR (shTAPBPR). Cells were radiolabelled for 60 min with [35S] cysteine/methionine, lysed, then equal aliquots of cleared lysates were either kept at 4°C or heated at 22°C, 37°C or 50°C for 12 min. Peptide loaded HLA-A2 was immunoprecipitated using BB7.2 post-preclear. After separation by SDS-PAGE, the signal intensity of the radiolabelled HLA-A2 band was determined by phosphorimaging. The results are representative of four independent experiments. The graph shows the percentage of peptide loaded HLA-A2 recoverable at each temperature as a percentage of the signal intensity at 4°C. Error bars show SEM from four independent experiments. Surface expression of (E) HLA-A2 (detected with BB7.2) and (F) HLA-B7 (detected with BB7.1) on cells treated with 5 µg/ml brefeldin A (BFA), which inhibits egress of newly assembled molecules from the ER, for 0, 0.5, 3, 6 and 16 hr. The level of remaining HLA-A2 and HLA-B7 at each time point is expressed as percentage of the mean fluorescence at time 0. Error bars represent SEM of duplicate samples and the data is representative of three independent experiments.