Fig. 1. Cellular distribution and nucleo-cytoplasmic shuttling of c-Myc. (A) c-Myc and c-Myc-GFP are predominantly nucleoplasmic. Both endogenous c-Myc (upper panels) and transiently transfected c-Myc-GFP (lower panels) were evenly distributed in the nucleoplasm while little or none was detected in the nucleoli of COS-7 cells. The cells (lower panel) were transfected with 1 µg of c-Myc-GFP expression plasmid. Bars, 20 µm. (B) FLIP analysis demonstrates that c-Myc-GFP shuttles between the nucleus and the cytoplasm. A COS-7 cell expressing c-Myc-GFP (outlined by the white dashed line) was repeatedly bleached, in a defined area in the cytoplasm (white circle). Confocal images were acquired before bleaching (pre-bleach) and between the bleach pulses. Images of the cell at the indicated times are shown. The lower cell, which was not bleached, serves as a control for unintentional bleaching during scanning. Bar, 20 µm. (C) Comparison of fluorescence loss in bleached and unbleached cells. The fluorescence loss curve indicates the mean value for relative fluorescence intensity measured in the nucleoplasm of cells subjected to FLIP, plotted as a function of time (filled squares). The error bars show standard deviation (n=5 cells). The filled triangles indicate the relative fluorescent intensity in the nucleoplasm of the neighbouring unbleached cell shown in B.