Chk1 depletion reduces Src-S51 phosphorylation. (A) Immunoprecipitation (IP) kinase assay using GST-FAK (871–1020) as substrate. Top: Western blot analysis of GFP-associated FAK-Y925 phosphorylation (pFAK-Y925) and immunoprecipitated GFP as well as Ponceau staining of GST-FAK (871–1020). Bottom: Western blot analysis of total GFP and actin. Relative band intensity values are shown, and values in GFP–Src-WT were set to 1. (B) Localization of phosphorylated Src-S51 (pSrc-S51). Cells were transfected with negative siRNA (control), siChk1, or siSrc. Broken DNA bridges are indicated by dotted arrows, and the bases of the intercellular canals are indicated by solid arrows. Insets show 1.6× magnification of the canals bases. Images are representative of 20 cells from two independent experiments. (C) Cells transfected as in B were seeded on fibronectin-coated slides for 1 h. Insets show 2× magnification of membrane ruffles. Bars, 5 µm. (D) Phosphorylated Src-S51 at membrane ruffles. Relative green fluorescence intensity from C is shown, and values in control were set to 1. Error bars show the SD from the mean. n = 20 cells from two independent experiments. ***, P < 0.001 compared with the control. Statistically significant differences were determined by ANOVA and Student’s t test. (E) Coimmunoprecipitation from asynchronous cells. Chk1 or Src were detected by Western blotting. Ab, antibody.