Nuclear AID is destabilized by ubiquitin-dependent proteolysis.

<p>(A) Representative examples of Ramos AID-mCherry transductants as analyzed by HCS, with whole cell boundary defined by HCS CellMask, yellow line; and nuclear boundary by DAPI, blue line. Typically, AID is cytoplasmic (N/C < 1), but treatment with LMB inhibits nuclear export of AID (N/C>1). In the examples shown, N/C = 0.80 (untreated) and 1.34 (0.5 hr LMB). (B) Scatter plots of nuclear vs. cytoplasmic mCherry (mCh) signals for untreated Ramos AID-mCherry transductants or for cells treated with MG132, LMB, or LMB+MG132 as indicated. (C) Quantification of total nuclear and cytoplasmic AID-mCherry signal by HCS, following treatment with MG132, LMB or LMB+Mg132 for indicated times. (D) Quantification of nuclear and cytoplasmic AID-mCherry signal and N/C ratio, relative to untreated cells, at indicated times post-treatment with MG132, LMB, or both. This analysis was repeated 3 more times for LMB treatment (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005411#pgen.1005411.s003" target="_blank">S3 Fig</a>), and once for MG132 and LMB+MG132 treatment. Dotted line represents no change (fold change of 1). Each point represents a population average, and black bars (too small to be discerned readily) represent SEM of the population.