Figure 4

Active focal adhesion kinase (FAK) associates with nucleoli in breast carcinoma cells. (A) Cell fractionation protocol for cytoplasmic, nucleoplasmic, and nucleolar isolation. (B) FAK pY397, total FAK, Lamin B, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and B23 immunoblotting analyses of FAK-WT and FAK-KD reconstituted MDA-MB-231 fractionation from adherent (0 h) and cells held in suspension for 4 h. FAK-WT and FAK-KD were detected within cytoplasmic and nucleoplasmic fractions. Greater FAK-WT levels and Y397 phosphorylated FAK were associated with purified nucleoli compared to FAK-KD. (C) Ratio of GFP-FAK to B23 levels determined by densitometry: means ± SD from three independent experiments with FAK-WT at 0 h set to 1 (*P <0.05, **P <0.01). (D) Representative confocal microscopy of green fluorescent protein (GFP)-FAK wild-type (WT) fluorescence and nuclei staining. Merge shows Hoechst (blue) and nucleolar GFP-FAK-WT (green) localization (arrows). Medial confocal section (scale 50 μm). (E) Representative pY397 FAK staining in paraffin-embedded normal and human breast carcinoma tumors. Normal breast contains little pY397 FAK signal; invasive ductal carcinoma (IDC) exhibits both cytoplasmic (right) and nuclear-associated anti-pY397 FAK staining (brown). Inset, higher magnification shows punctate pY397 FAK staining (arrows) within nuclei. Slides were counterstained with hematoxylin (scale 25 μm).