Figure 3
Focal adhesion kinase (FAK) is essential in mediating oncogenic signaling by transforming growth factor (TGF)-β. (a) Increased FAK expression correlates with TGF-β1-mediated activation of p38 MAPK in metastatic human breast cancer cells. The human MCF10A mammary carcinoma progression system (T1k, well differentiated; Ca1h, premalignant; and Ca1a, metastatic) was stimulated with TGF-β1 (5 ng/ml; tgf), or with TGF-β1 (5 ng/ml) in combination with epidermal growth factor (50 ng/ml; t/e) for 30 minutes and subsequently analyzed for phosphorylation of p38 MAPK (p-p38) or Smad2 (p-smad2), as indicated. Increased FAK expression was noted in metastatic Ca1a cells. Protein loading was monitored by reprobing stripped membranes with antibodies against total p38 MAPK (t-p38), total Smad2/3 (t-smad2/3), and β-actin. Data are representative images from a single experiment that was repeated twice. (b) Control (i.e., scram) and FAK-deficient (shFAK) 4T1 cells were stimulated with TGF-β1 (5 ng/ml) for varying times, as indicated. Afterward, cell extracts were prepared and immunoblotted with phospho-specific antibodies against Smad2 (p-smad2), Smad3 (p-smad3), or p38 MAPK (p-P38), as shown. Reprobing stripped membranes with antibodies against total p38 MAPK (t-p38), total Smad2/3 (t-smad2/3) FAK, and β-actin served as loading controls. Data are representative images from a single experiment that was repeated 3 times. (c) Control (i.e., scram) and FAK-deficient (shFAK) 4T1-luciferase cells were transfected with pSBE-renilla luciferase and stimulated with TGF-β1 (5 ng/ml) before measuring firefly and renilla luciferase activities. Data are expressed as the mean (± SEM) ratios of renilla:firefly luciferase activity observed in three independent experiments completed in triplicate. (*P < 0.05; **P < 0.0001). (d) Src phosphorylation (p-Src-Y416) was decreased in FAK-depleted (shFAK) 4T1 cells as compared with their control (i.e., scram) counterparts. Immunoblots were stripped and reprobed with antibodies against Src (t-Src), FAK, and β-actin as loading controls. Data are representative images from a single experiment that was repeated 3 times.