Deaminase activity and cytoplasmic localization of AID splice variants. (A) Dot plot of the somatic hypermutation assay depicting the frequency of GFP⁺ revertants in YFP⁺ NIH-3T3 cells. Each dot represents the measurement of one culture well. Virus stocks were applied in serial dilutions, resulting in a spread of the measurements (also visible in Figure 1B). (B) The same measurements shown in panel A depicted as a function of expression levels of the variants (mean IRES-YFP). Linear regression analysis shows the correlations between mutation frequency and expression levels. (C) In vitro deaminase activity: 100 fmol of a 60-nt FAM-labeled oligo, containing one cytidine located in a hot-spot motif, was incubated with 2 μg recombinant glutathione S-transferase (GST)–AID fusion protein. Subsequent recombinant UDG and NaOH heat treatments resulted in a 30-nt product, which was detected in AID-FL and T150A mutant, but not in the GST control, F151S AID mutant, or with any of the splice variants. Input and appropriate size of the recombinant proteins were verified by Coomassie staining (data not shown). (D) HEK293 cells were transfected with C-terminally tagged AID-GFP fusion proteins, and photographed alive at day 2 after transfection. AID-FL was located in the cytoplasm, but LMB incubation with 10 ng/mL for 3 hours resulted in nuclear accumulation. The AID splice variants display a predominantly nuclear or diffuse localization. Images were acquired with a Leica DM5000B microscope and a Leica DFC500 camera (Leica Microsystems, Rijswijk, The Netherlands) at the original magnification of 200×, and were further processed using Adobe Photoshop 7.