Regulation of fusion protein degradation by cell cycle. (A) Schematic of fusion proteins used in this study. The same symbols for degrons will be used in all figures. (B) Flow cytometric density plots of GFP fluorescence as a function of DNA content (DAPI fluorescence) in NIH-3T3 cultures in which all cells were transduced to express GFP-rag, mKO2-cdt, or GFP-cyc fusion proteins. Dashed lines indicate the approximate boundary of fluorescent and nonfluorescent populations according to nontransduced control cells. (C) The distribution of DNA content in subpopulations of double-transduced NIH-3T3 cells. Cells express mK02-cdt and GFP-cyc (left and middle) or mK02-cdt and GFP-rag (right). The left panel gates populations shown in the middle panel. (D) Images selected from time-lapse (15-min interval) confocal imaging of an NIH-3T3 cell double-transduced to express mKO2-cdt and GFP-cyc, which divided just before T = 0 min. Each image shows summed z-stacks for green and orange fluorescence overlaying a bright-field image. Immediately before cell division (T = −15 min) green fluorescence was intense. Fluorescence reverted to background upon cell division (T = 0 min), and no green or orange fluorescence was detectable above background in the daughter cells until T = 180 min, when orange fluorescence became detectable. (E) Anti-UNG2 Western blot of total protein extracts from 100% transduced 3T3 fibroblasts sorted into GFP⁺ and GFP⁻ populations (using gating identical to C). Samples were prestandardized to tubulin by semiquantitative Western blot. Experiments in all panels were performed at least twice.