Proteasome inhibition slows the degradation of BCR-associated ligand and delays the increased formation of ubiquitinated ligand–BCR complexes. (A) MD4.B10.Br splenocytes were pretreated for 1 hour at 37°C with or without 10 μM MG-132 as indicated. The cells were then pulsed with 10 μg/mL anti–IgM^a-btn for the indicated time (minutes) at 37°C in the continued presence of inhibitor. The ligand-pulsed cells were then washed and lysed, and the lysates cleared by centrifugation. The proteolytic degradation of the BCR-internalized anti–IgM^a-btn mAb in the whole-cell lysate was analyzed by reducing SDS-PAGE and Western blotting with SA-HRP (upper panel, ligand). The high-molecular-weight band (55 kDa, black arrowhead) represents the intact heavy chain of the anti–IgM^a-btn mAb. The prominent low-molecular-weight band (25 kDa, gray arrowhead) represents the intact light chain of the anti–IgM^a-btn mAb. The band of 30-kDa molecular weight (white arrowhead) is a proteolytic fragment of the heavy chain of the anti–IgM^a-btn mAb. Shown are representative results from 1 of 5 independent experiments. (B) A portion of the lysates analyzed in panel A was analyzed for the presence of anti–IgM^a-btn–BCR-ubiquitin complexes by precipitation of ubiquitinated proteins (including ubiquitinated ligand–BCR complexes) with p62 UBA–agarose and analysis of the samples by reducing SDS-PAGE and Western blotting with SA-HRP. The gray arrowhead marks the position of intact 55-kDa heavy chain of the anti–IgM^a-btn mAb, bound to the ubiquitinated BCR and thus precipitated by the p62 UBA–agarose. Shown are representative results from 1 of 3 independent experiments.