Western Blot Analysis of Amelogenin

Protein was extracted from gingival epithelial (G.E), CRUDE ERM, and the ERM clone cells as described previously^{50 (link)}. Briefly, protein samples (20 µg) were separated by electrophoresis on SDS–polyacrylamide precast gels (AnyKD Mini-PROTEAN, Bio-Rad; Hercules, CA, USA) and transferred to a polyvinylidene difluoride membrane (Immun-Blot PVDF membrane, Bio-Rad; Hercules, CA, USA). The membrane was incubated overnight with mouse monoclonal anti-amelogenin (1:500; Amelogenin [F-11], Santa Cruz Biotechnology; Dallas, Texas, USA) in Milli-Q water containing 0.2% Tween-20 and 4% skim milk at 4 °C. After washing, the membrane was incubated with appropriate horseradish peroxidase-conjugated goat anti-mouse IgG H&L (HRP) (1: 10,000; Abcam; Cambridge, UK) for 1 h at room temperature. Labeled protein bands were detected using an enhanced chemiluminescence system (LuminoGraph III, ATTO; Tokyo, Japan).