Subcellular targeting of GFP-LIMK1. (A) GFP-alone and three GFP-tagged LIMK1 proteins are depicted graphically and color coded. GFP was fused to the N-terminus of the LIMK1 cDNA. Exogenous NLS and NES tags were fused to the N-terminus of GFP. (B) Exogenous NLS and NES sequences target NLS-GFP-LIMK1 and NES-GFP-LIMK1 proteins to the nuclear and cytoplasmic subcellular compartments. Fluorescence microscopy was used to visualize subcellular localization of GFP fluorescence (green) in MDA-MB-231 cells. White-dashed lines are drawn to outline the nucleus of individual cells. Scale bar represents 20 microns. (C) Western blot analysis of cytoplasmic and nuclear fractions from MDA-MB-231 stable transductants. Nuclear segregation is assayed by total PARP. Cytoplasmic segregation is assayed by GAPDH. GFP-tagged LIMK1 is assayed with anti-LIMK1 antibody. Each lane is loaded total nuclear or cytoplasmic extract from 1 × 106 cells.