Mouse uPAR promotes VN-dependent p130Cas activation, cell spreading, and migration. (A) muPAR induces VN-dependent p130Cas substrate domain phosphorylation. Cells were incubated overnight in serum-supplemented medium in tissue culture dishes, washed, and lysed in SDS-PAGE sample buffer. Equal volumes of lysates were analyzed by western blotting using an antibody specific for p130Cas phosphorylated on Tyr410. The blots were stripped and then reprobed for VN as loading control. The lower panel shows the quantification of independent experiment (n = 5). (B) muPAR-induced cell spreading requires VN binding to the receptor. Cells in serum-supplemented medium were seeded sparsely on glass cover slides and left to adhere overnight. After fixation, DIC microscopy images were recorded and the cell-matrix contact areas quantified using ImageJ software. Each dot represents a single cell. Bars indicate the geometric mean with 95% confidence intervals. The data represent cell-matrix contact areas of randomly selected cells (n = 93–122) pooled from 2 independent experiments. (C) The interaction between uPAR and VN promotes migration of 293 cells on serum-coated surfaces. Cells grown in serum-supplemented medium were monitored by phase-contrast time-lapse microscopy. The migration speed of individual cells was quantified by manual cell tracking using the ImageJ software. Each dot represents the migration speed of a single cell. Bars indicate the geometric mean with 95% confidence intervals. The data represent migration speeds of randomly selected cells (n = 93–138) pooled from 2 independent experiments.