FIG. 1.

PTB relocalizes from the nucleus to the cytoplasm during adhesion. (A) MEFs were plated on fibronectin. After 24 h, the cells were fixed and immunostained with antibodies to PTB and vinculin. (B) MEFs were lifted, maintained in suspension for 1 h, and replated on fibronectin. Cells were allowed to adhere for 12 min (adhering) or 90 min (spread). Nonadhered cells were removed by washing, cells were fixed, and immunofluorescence was performed with antibodies to PTB and vinculin. The arrowhead shows PTB vinculin colocalization at a possible SIC. (C) MEFs adhered for 15 min were fixed and immunofluorescence performed with anti-PTB and antivinculin antibodies. Adhering cells were identified by an absence of focal adhesions (punctate vinculin staining in the cytoplasm), and spreading cells were identified by the presence of focal adhesions at the cell periphery. (D) The mean fluorescence intensity for PTB across the nucleus and the cytoplasm was determined for both adhering and spreading cells using Zeiss image analysis software. Cells were grouped as having a PTB cytoplasm-to-nucleus ratio of either >0.5, 0.5 to 0.3, or <0.3 (n = 56 for adhering and n = 47 for spreading). Magnification, ×40 (A) or ×63 (B and C). Scale bars, 20 μm.