Proteasome inhibition results in the accumulation of ubiquitinated proteins in splenic B cells. MD4.B10.Br splenocytes were incubated at 37°C for 1, 2, 3, or 5 hours (as indicated above each lane) in media containing either no drug, 10 μM MG-132, or 10 μM lactacystin. The presence of ubiquitinated proteins in the cell lysates was determined by reducing SDS-PAGE and Western blotting with the 6C1 antiubiquitin mAb. The faint band at approximately 24 kDa is the light chain of the endogenous BCR, as it was detected in blots probed with secondary antibody alone (not shown). The prominent band of approximately 60-kDa molecular weight is a constitutively ubiquitinated protein (as opposed to detection of the endogenous BCR), as this band was not observed in blots probed with secondary antibody only. Similar results were obtained whether or not the B cells had been stimulated via the BCR (data not shown). Shown are representative results from 1 of 3 independent experiments.