Cathepsin k

BMDMs were incubated with 30% of L929-conditioned medium and 100 ng/mL of RANKL with or without ChondroT or its constituent herbs for indicated time. Cell lysates were quantified with Bradford’s reagent (Bio-Rad, USA). Equal concentrations of proteins were subjected to 10% SDS-PAGE, and were transferred onto PVDF membranes (Millipore Ltd., Carrigtwohill, Germany). After blocking, membranes were incubated with antibodies specific to osteoclast related proteins (c-Fos, NFATc1, MMP9, Cathepsin K, GAPDH, 1:1000, Santa Cruz, CA, USA), MAPKs signals [JNK (1:500, Santa Cruz, CA, USA) and (p-JNK, ERK, pERK, p38, p-p38, 1:500, Cell signaling Tech, MA, USA)], and NF-κB signals (NF-κB p65, LaminB, β-actin, 1:1000, Santa Cruz, CA, USA), and then incubated with HRP-conjugated secondary antibodies. Immunoreactive proteins were detected with an ECL Western blot detection system (Advansta, Menlo Park, CA, USA).

Maxillae tissue was deparaffinized and rehydrated. Gingival and periodontal tissue slices (4 μm) were washed with 0.3% Triton X-100 in phosphate buffer, quenched with endogenous peroxidase (3% hydrogen peroxide), and incubated overnight at 4°C with primary antibodies against the following proteins (all antibodies 1:400): receptor activator of the NF-κB ligand (RANKL), superoxide dismutase-1 (SOD-1), receptor activator of NF-κB (RANK), glutathione peroxidase-1 (GPx-1), osteoprotegerin (OPG), matrix metalloproteinase 2 (MMP-2), cathepsin K, and cyclooxygenase-2 (Santa Cruz Biotechnology, INTERPRISE, São Paulo, SP, Brazil) and incubated for 30 min with a streptavidin/horseradish peroxidase-conjugated secondary antibody (Biocare Medical, Concord, CA, USA). Immunostaining was visualized using colorimetric detection (Biocare Medical, Dakota, USA). The staining status was identified as either negative or positive; positive staining was defined as the presence of brown chromogen. Staining intensity and the proportion of immunopositive cells were examined independently by two pathologists by light microscopy and recorded. Intensity of staining (IS) was graded on a 0 to 2 scale according to the following semi-quantitative assessment: 0 = no detectable staining, 1 = weak staining; 2 = strong staining.²²

Radioimmunoprecipitation assay (RIPA) lysis buffer (Millipore Sigma, Burlington, MA) was prepared by mixing RIPA with protease inhibitor and phosphatase inhibitor (1:100). The medium was aspirated from the wells and the cells were washed 3x with 5 mL PBS. A volume of 300 μl RIPA buffer was added to each well. The cells were scraped off the wells and transferred to 1.5 mL tubes incubated on ice. The cells were sonicated on ice and centrifuged at 4°C for 10 minutes at 13,200 rpm. The supernatant was transferred to separate tubes and placed on ice for PAGE or stored in the -80^o freezer. Antibodies used for this study included1^o Ab specific to p-STAT3 (Ser727), NFATc1, c-Fos, and Cathepsin K; all were purchased from Santa Cruz Biotechnology (Dallas, Tx); 1^o Ab specific to ß-actin Ab was obtained from Sigma-Aldrich, (St Louis, MO).

Fetal bovine serums (FBS), α-modified essential medium (α-MEM), and penicillin were purchased from Gibco (Gaithersburg, MD, USA). RANKL was purchased from PeproTech (Rocky Hill, NJ, USA), and M-CSF was purchased from R&D Systems (Minneapolis, MN, USA). All primary antibodies as phospho-p38, p38, phospho-ERK, ERK, phospho-JNK, JNK, phospho-Akt, Akt, and c-Fos were obtained from Cell signaling Technology (Danvers, MA, USA). NFATc1 and cathepsin K were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Trizol and Supercript II Reverse Transcriptase were purchased from Invitrogen (Carlsbad, CA, USA). Calcium phosphate (CaP) coated plates were purchased from Cosmo Bio (Tokyo, Japan). All other chemicals were purchased from Sigma (St. Louis, MO, USA) unless otherwise indicated [24 (link)]. RT-PCR primers are listed in Table 1.