Figure 3. FAK is activated downstream of ROCK-mediated actomyosin contraction during SMG branching morphogenesis.

(A) E13 SMGs were cultured for 24 hours with increasing concentrations of Y27632 or blebbistatin added to the culture media, and cell lysates were immunoblotted with antibodies specific for FAK phosphorylated on Tyr 397 or Tyr 576/577. We observed a dose-dependent decrease in FAK phosphorylation at both of these sites, with quantification shown relative to total FAK protein.

(B) E13 mesenchyme-free epithelial rudiments cultured with vehicle control, Y27632, or blebbistatin were immunostained for FAK phosphorylated on Tyr 397 (green) and the focal adhesion protein talin (red). In vehicle control rudiments, pFAK Y397 localized to the periphery of epithelial buds (white arrows) with the focal adhesion protein talin. Both pFAK Y397 and talin localization and staining were reduced with the ROCK and myosin inhibitors. Scale bar, 10 μm (B).