Fig. 2.

Normal nucleocytoplasmic shuttling of AID R-mutants. a (Left) Illustration of the mechanisms regulating AID nucleocytoplasmic shuttling. Didemnin B (Did B) inhibits cytoplasmic retention (CR); leptomycin B (LMB) inhibits nuclear export (NE). (Right) Representative confocal microscopy images of AID-deficient CH12 cells transduced with untagged AID variants and analysed by immunofluorescence with anti-AID antibody. Cells were treated for 2 h with DMSO or 10 ng mL⁻¹ LMB and/or 100 nM Did B. (Bottom) Proportion of nuclear AID signal for individual cells (dots, n indicated above each group). Horizontal bars are mean values. The dotted blue line indicates the mean value of untreated cells expressing wt AID. Compilation of two independent experiments. b Representative WBs on cytoplasmic or nuclear lysates from reconstituted AID-deficient CH12 cells probed with anti-AID, -Gapdh and -LaminB antibodies. For gel source data, see supplementary Fig. 7. c Representative confocal microscopic images of complemented AID-deficient CH12 cells analysed using anti-AID and anti-Spt5. Cells were determined to be cycling (G1/S/G2), mitotic or in cytokinesis based on DNA condensation and Spt5 access to the DNA. Images are representative of 10–12 observed events per construct from 2 independent experiments. a, c Magnification 630×. Scale bar, 10 μm