Figure 1.

Analytical methods. Serum samples obtained by centrifugation at 3000 × g for 15 minutes at room temperature are centrifuged again for 2 hours at 16,000 × g at 4°C. Levels of serum or supernatant fetuin-A are measured using ELISA. Supernatants are discarded, then centrifuge tubes are rinsed twice with 150 mM NaCl and re-centrifuged. Resultant pellets are dissolved in 150 mM HCl for calcium (Ca) analysis. Pellets are dissolved in Laemmli buffer containing 60 mM EDTA and resolved by Super-Sep PAGE. Proteins of interest are identified by Coomassie Brilliant Blue (CBB) staining and MALDI-TOF/MS.