Proteasome inhibition delays the dissociation of internalized antigen-BCR complexes. (A) MD4.B10.Br splenocytes were pretreated with proteasome inhibitor and pulsed with antigen as described in “Materials and methods.” The cells were then fixed and stained for BCR-dissociated antigen, using the HyHEL10 monoclonal antibody that recognizes the same HEL epitope as the MD4 BCR (see image at the top of the figure and Gondré-Lewis et al¹¹ ). The cells were then analyzed by fluorescence microscopy, and the percent of B cells containing detectable BCR-dissociated antigen was determined. The results demonstrate that while BCR-dissociated antigen is readily detectable 4 hours after internalization under all conditions, BCR-dissociated antigen is present for a prolonged period of time in proteasome-inhibited B cells. Shown are average values from 2 independent experiments (bars). The error bars indicate the range of values obtained across both experiments. (B) B10.Br splenocytes were treated as indicated (no drug or 10 μM MG-132, lactacystin, or brefeldin A), and then allowed to internalize HEL by fluid-phase endocytosis for 30 minutes. The cells were then washed, chased for the indicated times, fixed, and stained for HEL and the LE/L marker LAMP. The percent of B cells in which HEL was detectable within LAMP⁺ LE/L is shown. The results demonstrate that unlike treatment with brefeldin A (which is known to inhibit trafficking between the earlier and later aspects of the endocytic pathway), treatment of the cells with the proteasome inhibitor fails to slow the delivery of fluid-phase markers to the later aspects of the endocytic pathway. It should be noted that at the 6-hour time point, the B cells contained low overall levels of detectable HEL. Shown are representative results from 1 of 3 independent experiments.