Doxycycline induces FAK and FAK-CD in MCF-7-Tet-ON cell line. A, B, MCF-7-Tet-ON stable cell line was generated with stable expression of FAK (A) or FAK-CD (B). Addition of doxycycline (2 μg/ml) for 6 days resulted in overexpression of FAK or FAK-CD. Western blotting with HA-tag antibody was performed to detect HA-tagged FAK. Western blotting with Y397 antibody shows expression of activated FAK in MCF-7-Tet-ON-FAK cells. Western blotting with 118-Y-paxillin antibody demonstrates increased phosphorylation of FAK-substrate paxillin in MCF-7-Tet-ON-FAK cells. Western blotting with beta-actin shows equal loading of proteins. C, Exogenous FAK localizes to focal adhesions. MCF-7-Tet-ON-HA-FAK or control MCF-7-Tet-TRE-2 cells. Immunostaining with HA-tag antibody was performed to detect HA-tagged FAK. Staining with FITC-conjugated phalloidin detected actin in the cells. Dox, doxycyclin. D, Cell rounding and displacement of FAK from the focal adhesions in MCF-7-Tet-ON-FAK-CD cells. To detect FAK in the cells, we stained cells for FAK with 4.47 N-terminal FAK antibody. Upper panels: MCF-7-Tet-ON-TRE-2 cells, lower panels: MCF-7-Tet-ON-FAKCD cells.