Las 4000 ccd imaging system

A previously described method was used for western blot analysis [27 ]. In brief, total proteins were extracted from the MC3T3 E1 cells with a lysis buffer containing 150 mM NaCl, 5 mM EDTA, 50 mM Tri-HCl (pH 8.0), 1% NP 40, 1 mM pepstatin, 1 mM aprotinin, and 0.1 mM leupeptin. Proteins in the cells were quantified by the Bradford dye-binding procedure (Bio-Rad, Hercules, CA, USA). The samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8% to 15%) under denaturing conditions and transferred to a Hybond-P membrane (Amersham, Arlington, IL, USA). Specific primary antibodies were used at a ratio of 1:1,000 to 1:2,000 and incubated at 4°C overnight, and then incubated with a horseradish peroxidase-IgG-conjugated secondary antibody at room temperature for 1 hour. A chemiluminescence detection reagent was used to detect the signals according to the manufacturer's protocol (Amersham Pharmacia Biotech, London, UK) with a LAS-4000 CCD imaging system (Fujifilm, Tokyo, Japan).