Fig. 4.

The RR domain is dispensable for AID association with tightly held nuclear complexes. a Representative confocal microscopic images of CH12 B cells either fixed directly (whole cell) or after nuclear washing (0.5% Triton+200 mM NaCl). Isolated nuclei were analysed by IF to detect endogenous AID and Spt5 and DNA stained by Dapi. Cells were stimulated (+CIT) to induce AID expression or not (−CIT). Representative of three experiments. b (Left) Representative confocal microscopic images of GFP, AID (detected by IF) and DNA (Dapi) in reconstituted AID-deficient CH12 B cells, imaged as in a. AID and GFP expression were linked via IRES. (Right) Nuclear AID signal was calculated relative to whole-cell intensity for each variant and normalized to the wt AID value. Plotted are means (bars) from 3 independent experiments (dots) with 20–77 cells per condition per experiment. a, b Magnification 630×. Scale bar, 10 μm. Laser power and/or gain were increased for imaging after nuclear wash (see Methods). c Scheme for biochemical fractionation of B cells. Fractions analysed are  indicated in brackets. d Representative WBs on the indicated fractions from reconstituted AID-deficient CH12 B cells. Antibodies against AID, RNAPII, Spt5, Lamin B and Gapdh were used as indicated. Representative agarose gel with purified DNA stained using ethidium bromide and Ponceau-S staining of total protein are also shown. For gel source data, see supplementary Fig. 7. e Quantification of AID signal in each lane from d, normalized to the respective cytoplasmic AID. Means + s.d. from three independent experiments