Figure 7.
eEF1A, CRM1, and Hsp90 associate with functionally distinct AID fractions. (A) Representative confocal microscopy images are shown of endogenous AID (detected by IF) in Ramos B cells (left). Cells were treated for 2 h with DMSO, 2.5 µM CHX, 100 nM DidB, or 1 µM CytA in combination with either vehicle or 10 ng/ml LMB. Scale bar, 10 µm. The proportion of nuclear AID signal for ≥100 cells per point was assessed as the ratio of AID signal overlapping DAPI signal to total AID signal × 100. The nuclear AID signal of each cell (dots), and the mean for each group (black bars) are shown for one out of 2 independent experiments, (right graph). (B) Ramos B cells were treated as in (A) for 6 h and expression of AID, actin and eEF1A were analyzed by Western blot. One representative out of 2, independent experiments is shown. The mean ratio of AID to actin measured by densitometry, normalized to DMSO is indicated under the relevant blots. (C) Ramos B cells stably expressing either GFP or human AID-GFP were treated in duplicate as in (A). The MFI ± SEM of GFP was measured by flow cytometry at different times post-treatment and normalized to t0 = 100%, and was plotted over time for one out of ≥2 representative, independent experiments. (D) Ramos B cells stably expressing AID-GFP were treated for 24 h, as indicated, and the nuclear GFP signal was quantified as in A (top). The MFI + SD of AID-GFP was measured by flow cytometry and normalized to vehicle, and was plotted from 4 independent experiments (bottom). Means were compared by Student’s t test (*, P < 0.05; **, P < 0.005). (E and F) Representative plots of the proportion of IgG1+ cells per division are shown below the corresponding CFSE profiles in wt mouse naive splenic B cells stimulated with 5 µg/ml LPS and 5 ng/ml IL-4 in the presence of the indicated inhibitors 24 h later. The proportions of IgG1+ cells were determined by flow cytometry 3 d after stimulation (shown in bottom graphs). Data were compiled from 4 mice from 2 experiments, (n = 2 mice/experiment). Differences relative to vehicle were analyzed by two-way ANOVA (*, P < 0.05; **, P < 0.005; ****, P < 0.0001).