Phosphorylation of p130Cas does not depend on actomyosin contractility.

<p>(A) Scheme showing a cell experiencing a tensile force from the extracellular matrix (ECM). (B) Close-up view of the molecular interactions occurring at the cell–ECM junction. Proteins associated with the adhesions (multi-colored circles) and a mechanosensing protein (yellow) are shown as stretched by the inward-facing cytoskeletal contraction forces (<i>F</i>_Cytoskeletal) and the external tensile forces (<i>F</i>_External). (C) p130Cas-deficient fibroblasts expressing GFP–p130Cas in the presence (the “Blebbistatin” row) or absence (the “DMSO” row) of 10 µM blebbistatin were viewed with a TIRF microscope. Differential interference contrast (DIC, first column), GFP TIRF (Cas, second column), and Alexa546 TIRF (p-Cas, third column) images of a leading cell in scratched monolayer were acquired. Merged images of GFP and Alexa546 (Merge, fourth column) are shown. Scale bar: 10 µm. (D) HEK293 cells cultured on collagen-coated substrate were treated with DMSO (0.1%), blebbistatin (50 µM), cytochalasin D (0.5 µM) or latrunculin B (0.5 µM) for 30 minutes. After treatment, the cells were lysed and equivalent amounts of cell lysates were subjected to SDS-PAGE followed by western blot analysis using anti-phospho-p130Cas-Y165 (pCas-165) and anti-p130Cas (αCas3) antibody.