Quantifying Gingival MMP and RANKL

Protein levels of matrix metalloprotease (MMP) and receptor activator of nuclear factor-κB ligand (RANKL) were assessed by WB analysis. The gingival tissue around the maxillary second molar was carefully removed and homogenized. After quantitative determination of protein concentration with the tissue BCA method, we executed WB analysis of the target protein and internal reference protein as previously described [27 (link)]. The exposed film was scanned directly, and the image format was converted with Image J software (NIH, Bethesda, MD, USA). The integrated optical density (IOD) values of the bands were read and analyzed with Total Lab Quant V11.5 (Newcastle upon Tyne, UK). Primary antibodies against β-actin, MMP-8, MMP-9, and RANKL were purchased from Abcam (Cambridge, MA, USA).