Chk1-inhibition reduces Src kinase activity. (A and B) Cells transfected with negative siRNA (control) or siChk1 were trypsinized and seeded on fibronectin-coated dishes in the absence or presence of PP2 for 1 h. Western blot analysis of total phospho–Src-Y419 (pSrc-Y419), Src, phospho–FAK-Y925 (pFAK-Y925), FAK, or actin. NS, nonspecific. Relative band intensity values are shown, and values in control were set to 1. (C) Chk1 depletion reduces cell spreading. Cells were seeded on fibronectin-coated dishes and phase-contrast images taken at various times after plating. Bars, 50 µm. (D) Percentage of spread cells (i.e., flattened and not highly reflective) from C. Error bars show the SD from the mean from three independent experiments. Approximately 750–1,000 cells were analyzed per experiment. (E) Cells were seeded on fibronectin-coated dishes and analyzed by wound healing assay. Phase-contrast images at 0 h and 12 h after wounding are shown. Bars, 200 µm. (F) Wound area covered calculated from cells as in E. Error bars show the SD from the mean from four independent experiments. **, P < 0.01; ***, P < 0.001 compared with the control. Statistically significant differences were determined by ANOVA and Student’s t test.