Lysine mutants of AID are polyubiquitinated in BL2. (A) Schematic presentation of the human AID protein sequence along with the positions of the lysine residues mutated in the various constructs. (B) BL2 clones expressing either WT-AID-EGFP (sample 1, WT), or a lysineless mutant of AID (Kzero-AID-EGFP; sample 2, Kzero) were incubated with the indicated inhibitors for 5 h. Protein extracts were analyzed by immunoblotting with anti-AID. PARP (116 kD) was used as loading control. (C) BL2 cell clones stably expressing either WT-AID-EGFP (WT), mutNES-AID-EGFP (mutNES), a lysineless mutant of AID (Kzero), or the four other AID-EGFP lysine mutants depicted in A were transiently transfected with an HA-tagged ubiquitin-expressing vector and either left untreated (top) or incubated with both LMB and MG132 for 5 h (bottom). Denatured lysates were immunoprecipitated with agarose-conjugated anti-HA antibodies and analyzed by Western blotting using anti-EGFP antibody. Migration position of AID-EGFP is indicated. (D) Stable BL2 clones expressing the indicated AID-EGFP lysine mutants, WT-AID-EGFP, or mutNLS-AID-EGFP were incubated with cycloheximide (CHX, 50 μg/ml) and LMB (10 ng/ml). The decay of the protein was followed by the decrease in fluorescence intensity. The percentage of initial fluorescence is plotted at various time points after addition of the drugs. Kzero1 and 2 represent two independent clones. (top) Clones transfected with AID constructs cloned in the pIRES puro expression vector; (bottom) clones transfected with tetracycline-inducible pBI expression vectors.