Table II.

Localization of Injected Zyxin-GST Fusion Proteins in Rat Embryo Fibroblast (REF-52) Cells

Construct		Nucleus (percentage of cells)		Cytoplasm (percentage of cells)		Number of cells
GST		100		0		130
GST-Zyx305-348		20		80		36
GST-Zyx319-335		15		85		120
GST-Zyx322-331		100		0		115

To test the ability of zyxin's conserved, leucine-rich region to relocalize nuclear proteins to the cytoplasm, the distribution of nuclear injected GST, with and without fused zyxin aa sequences, was determined by immunofluorescence. GST or GST zyxin fusion proteins were expressed and purified from bacteria and co-injected into REF-52 cell nuclei along with fluorochrome-labeled BSA to mark successful nuclear injection. Within 30 to 45 min of injection, cells were prepared for indirect immunofluorescence using an antibody directed against the FLAG epitope tag found within the GST leader peptide. The percentage of cells that displayed nuclear or cytoplasmic localization of the fusion protein is shown; three independent experiments were performed.