Protein Expression Analysis in Enamel Development

Western immunoblotting was performed, as we previously described [23 (link)]. Briefly, total proteins were extracted from the enamel organ tissues of the mandibular incisors of 3-day-old Fam20C^fl/fl and Sox2-Cre;Fam20C^fl/fl mice. Western immunoblotting was then performed to detect BMP4, P-Smad1/5/8 and P21 using the corresponding antibodies described above. The secondary antibodies used were horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology; 1:1000) or HRP-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology; 1:1000). β-actin was immunoblotted with mouse monoclonal anti-β-actin-peroxidase antibody (Sigma; 1:20,000). The immunostained protein bands were detected with ECL™ Chemiluminescent Detection reagents (Amersham Biosciences, Illinois, USA) and imaged using a CL-XPosure film (Pierce Biotechnology, Inc., New Jersey, USA).