Fibronectin

Cells were lysed in PRO-PREP Protein Extraction Solution (iNtRON Biotechnology, Inc., Gyeonggi-do, Korea) to prepare protein lysates according to the manufacturer’s instructions. Cell lysates were centrifuged at 12,000 rpm for 10 min at 4 °C. Protein concentration was measured using Bradford Easy Protein Quantitative Kit (TransGene). Equal amounts of protein were separated in 10% polyacrylamide gels (Sigma-Aldrich) and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 10% non-fat milk and incubated with anti-α-SMA (Abcam, Cambridge, UK), anti-collagen I (Abcam), anti-E-cadherin (Abcam), anti-N-cadherin (Bioss, Beijing, China), albumin (Bioss), matrix metalloproteinase 9 (MMP9) (Bioss), vimentin (Bioss), pan-cytokeratin (a wide spectrum cytokeratin antibody, Bioss), fibronectin (Bioss), β-catenin (Sigma) and anti-GAPDH (TransGene) antibodies at 4 °C overnight. After washing the membranes with Tris-buffered saline containing Tween 20 three times for 5 min each time, the membranes were incubated with secondary antibodies. Finally, immunoreactive proteins were detected using an enhanced chemiluminescence detection kit (Beyotime, Shanghai, China).

Mouse monoclonal anti-actin and anti-TRPC4 were purchased from Abcam (Cambridge, MA) and Antibodies Inc. (Davis, CA), respectively. Rabbit polyclonal anti-CaMKII, -pCaMKII, and -pCREB were purchased from Cell Signaling Technology (Danvers, MA). Rabbit polyclonal anti-collagen IV and -fibronectin were obtained from Bioss Inc. (Woburn, MA). HRP-conjugated anti-rabbit and anti-mouse secondary antibodies were purchased from Thermo Scientific (Waltham, MA) and Abcam, respectively. Cross-adsorbed Alexa Fluor 555 goat anti-rabbit was purchased from Life Technologies (Grand Island, NY). Fura-2 AM, Pluronic F-127, mouse UII, urantide, β-mercaptoethanol, SGC-CBP30, and Pyr3 were purchased from Life Technologies, AnaSpec (Fremont, CA), Phoenix Pharmaceuticals (Burlingame, CA), Peptides International, Inc. (Louisville, Kentucky), Bio-Rad, Tocris Bioscience (Bristol UK), and Cayman Chemical (Ann Arbor, MI), respectively.