International Journal of Cell Biology / 2014 / Article / Fig 1

Research Article

Matrix Gla Protein Binds to Fibronectin and Enhances Cell Attachment and Spreading on Fibronectin

Figure 1

MGP binds to fibronectin and to the first type III repeat domain of fibronectin III1-C. (a) MGP binds fibronectin and fibronectin fragments with type III repeats. Dot blot spots with radioactive MGP probe and 100 microliters of protein solutions were dot blotted onto the nitrocellulose filter, then blocked with 1% BSA-PBS, overlaid with radioactive MGP, washed, and exposed to film as described in methods. C, PBS buffer; OA, 1 mg/mL ovalbumin; FNIII1C, 1 mg/mL fibronectin III1-C; 30 KFN, 1 mg/mL 30 kDA fragment of fibronectin; 45 KFN, 1 mg/mL 45 kDA fragment of fibronectin; 110 KFN, 1 mg/mL 110 kDA fragment of fibronectin; 1991–1997, 1 mg/mL of the cell binding peptide of fibronectin comprised of amino acids 1991 through 1997; FN, 1 mg/mL fibronectin; anti-MGP, 5 μg/mL of anti-MGP IgG from rabbit. The lower row of dot blotted proteins contains 10x lower concentration of proteins. (b) Radioactive MGP probe binds to fibronectin III1-C on far western blot. Fibronectin III1-C is run on SDS-PAGE, electroblotted, blocked and overlaid with iodinated MGP, washed, and exposed to film as described in methods. (c) Both MGP and the 61–77 C-terminal peptide of MGP bind to fibronectin III1-C. Far western blot with either 125I-MGP or overlay on fibronectin III1-C and exposed to film as described. Both probes bind to a band that comigrates with a gel band of fibronectin III1-C shown stained with amido black next to molecular weight markers.