Acute proteasome inhibition selectively inhibits BCR-mediated antigen processing and presentation. B cells were pretreated with the indicated drug for 1 hour at 37°C. The cells were then pulsed with antigen (BCR indicates MD4.B10.Br pulsed with 100 nM HEL; F-P, B10.Br pulsed with 100 μM HEL and 100 nM anti–murine IgM antibody) for 4 hours in the continued presence of drug before washing and an additional 18 to 20 hours of incubation at 37°C in the absence of drug or antigen. The cells were then harvested and the level of HEL_46-61–I-A^k complexes expressed on the surface of the cell was determined by staining with the HEL_46-61–I-A^k complex–specific mAb C4H3 and subsequent analysis by flow cytometry.^5,11,14  Experiments performed with lactacystin gave similar results to those obtained with MG-132. However, this treatment resulted in a significantly greater level of B-cell death, presumably due to the irreversible nature of the inhibitor (data not shown). Parallel analysis of each sample for the expression of total I-A^k class II molecules (by staining with the panreactive mAb 11-5.2) revealed that none of the drug treatments resulted in a significant (ie, > 10%) decrease in the level of total I-A^k molecules expressed by the cells. Consistent with previous reports that HEL_46-61 is presented exclusively on newly synthesized I-A^k molecules,^5,14  treatment with either 10 μM puromycin or 10 μg/mL brefeldin A inhibited C4H3 binding by 80% to 90% under all conditions. The bars indicate the average level of HEL_46-61–I-A^k complexes expressed under the indicated conditions, as a percent of the level observed in non–drug-treated cells. The error bars indicate the range of experimental values obtained under each condition. Shown are from results from 2 independent experiments.