Over expression of ZAR2 protein and its subcellular location in MCF7 and MDA-MB-231 cells. (A) RT-PCR analysis showing the expression of N-terminal (i) and C-terminal (ii) FLAG-tagged ZAR2 mRNA in MCF7 cells. ZAR2-1 and ZAR2-2 are two independently derived transfectants. Similar results were obtained with MDA-MB-231 cells. (B) Western blotting analysis with anti-FLAG antibody showing expression of N-terminal (i) and C-terminal (ii) FLAG-tagged ZAR2 protein in MCF7 cells. Similar results were obtained with MDA-MB-231 cells. (C) Immunofluorescence analysis showing predominantly cytosolic location of N-terminal FLAG-tagged ZAR2 protein in the dividing MDA-MB-231 cells (left panel); and the C-terminal FLAG-tagged ZAR2 in dividing MCF7 cells (right panel). Anti-FLAG M2 antibody was used for the detection of FLAG-tagged ZAR2 protein in the cells. The cells were transiently transfected with the expression plasmid constructs and thus not all cells are expressing the recombinant protein. (D) Immunofluorescence confocal microscopy after dual labeling of the unsynchronized C-terminal FLAG-tagged ZAR2-expressing MCF7 cells with reagents for FLAG-ZAR2 (red) and the S-phase marker cyclin A (green). Predominant levels of FLAG-ZAR2 in the cytosol of the cells that have high levels of cyclin A in the nucleus. Nucleus was stained with Topro for confocal microscopy.