Chemiluminescent detection reagents ecl

Western immunoblotting was performed, as we previously described (Liang et al. 2016 (link); Wang et al. 2010 (link)). For Western immunoblotting analyses of DMP1 and DSP/DSPP, total proteins were extracted from the maxillary and mandibular first molars of the 3-week-old Fam20a^fl/fl and Sox2-Cre;Fam20a^fl/fl mice, as described in our previous publications (Liang et al. 2019 ; Qin et al. 2001 (link); Sun et al. 2010 (link)). For Western immunoblotting analysis of FAM20C, total proteins were extracted from the enamel organ tissues of the mandibular incisors of 3-day-old Fam20a^fl/fl and Sox2-Cre;Fam20a^fl/fl mice. Western immunoblotting was then performed to detect DMP1 and DSP/DSPP using rabbit polyclonal antibodies described above, and FAM20C using a FAM20C rabbit polyclonal antibody, as previously described (Wang et al. 2010 (link)). The secondary antibodies were horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology; 1:1000). β-actin was immunoblotted with mouse monoclonal anti-β-actin-peroxidase antibody (Sigma; 1:20,000). The immunoreactive protein bands were visualized with ECL™ Chemiluminescent Detection reagents (Amersham Biosciences, Illinois, USA) and imaged using a CL-XPosure film (Pierce Biotechnology, Inc., New Jersey, USA). Three independent mice were analyzed for each genotype and antibody, and one representative experiment was shown.