Focal adhesion kinase (FAK) is critically involved in normal mammary epithelial cell (MEC) migration and p38 MAPK signaling stimulated by transforming growth factor (TGF)-β. (a) Control (i.e., scram) and FAK-deficient (shFAK) NMuMG cells were stimulated with TGF-β1 (5 ng/ml) for varying times as indicated, at which point whole-cell extracts were prepared and immunoblotted with phospho-specific antibodies against Smad2 (p-smad2), Smad3 (p-smad3), or p38 MAPK (p-p38) as shown. Immunoblot analysis of total p38 MAPK, Smad2/3, FAK, and β-actin serves as loading controls. Data are representative images from a single experiment that was repeated 3 times. (b) Control (i.e., scram) and FAK-deficient (shFAK) NMuMG cells were transiently transfected with pSBE-luciferase and pCMV-β-gal cDNAs, and subsequently were stimulated with TGF-β1 (5 ng/ml) for 18 hours. Afterward, luciferase and β-gal activities present in detergent-solubilized cell extracts were measured. Data are expressed as the mean (± SEM) ratios of luciferase:β-gal activity observed in three independent experiments completed in triplicate. (c) Fluorescently labeled phalloidin staining showed the formation of F-actin stress fibers in control (i.e., scram) NMuMG cells treated for 18 hours with TGF-β1 (5 ng/ml). FAK-deficiency (shFAK) significantly impaired the ability of TGF-β1 to induce stress fibers, as well as disrupted normal actin cytoskeletal architecture. Data are representative images from a single experiment that was repeated twice. (d) Control (i.e., scram) and FAK-deficient (shFAK) NMuMG cell migration into denuded wounds over a 48-hour period in the absence (NS) or presence of either the TβR-I inhibitor, SB-431542 (10 μM, SB) or TGF-β1 (5 ng/ml). Data depict the mean (± SEM) percentage of wound closure measured in five independent experiments. FAK deficiency inhibited tonic and TGF-β1-stimulated NMuMG cell migration. In addition, tonic NMuMG cell migration was inhibited by SB-431542, indicating disruption of autocrine TGF-β signaling in wounded cultures (*P < 0.05; **P < 0.005).