Anti cathepsin k

The left femur was fixed in 10% NBF for 2 days, demineralized using 10% EDTA-2Na for 3 weeks, and then dehydrated with ethanol, clarified with xylene, and embedded with paraffin. Paraffin embedded tissue was sectioned on a rotary microtome. Endogenous peroxidase was blocked in 3% H₂O₂/Mt-OH for 15 min at room temperature. Then, 20 μg/ml proteinase K (Thermo fisher, PA, USA) was used for epitope retrieval for 10 min at 37 °C, for blocking, normal serum (Gibco, Gaithersburg, MD, USA) was reacted at room temperature for 30 min. The tissues were incubated with primary antibody diluted in 0.5% BSA, including anti-NFATc1 (1:100, Santa Cruz Biotechnology, CA, USA), anti-c-fos (1:100, Santa Cruz Biotechnology, CA, USA) and anti-cathepsin K (1:100, Santa Cruz Biotechnology, CA, USA), at 4 °C overnight. The tissues were then stored in 1:100 biotinylated secondary antibody (Vector Labs, Burlingame, CA, USA) for 60 min at room temperature. The tissues were incubated in horseradish peroxidase-streptavidin using ABC kit (Vector Labs, Burlingame, CA, USA) for 30 min at room temperature and stained with 3,3′-Diaminobenzidine solution (Vector Labs). The tissues were counterstained with hematoxylin, dehydrated and mounted. The immunohistochemical stained tissues were observed with a light microscope (100, 200×).

Total proteins were obtained by using radio immunoprecipitation assay (RIPA) buffer (Thermo Scientific, Waltham, MA, USA) containing a protease inhibitor cocktail (protease inhibitor cocktail 1 tablet; Roche Diagnostics, Mannheim, Germany) and centrifuged at 13,000 rpm for 10 min. The protein was measured with BCA Protein Assay Kit by using microplate reader at 562 nm. Equal amounts of protein were separated on 10% SDS-PAGE gel electrophoresis and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA). After blocking with 5% bovine serum albumin, protein was probed with appropriate antibodies.
The primary antibodies used were as follows: anti-TRAP, anti-cathepsin K, anti-c-Fos, anti-NFATc1, anti-TRAF6, and anti-β-actin from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-carbonic anhydrase II, anti-IκBα, and anti-phospho-IκBα from Abcam (Cambridge, MA, USA); and anti-IL-1β and anti-cleaved IL-1β from Cell Signaling Technology (Danvers, MA, USA). The SuperSignal® West Pico chemiluminescent kit (Thermo Scientific, Rockford, IL, USA) was used to detect the target protein. Images were obtained using a ChemiDoc TM XRS system (Bio-Rad Laboratories, Hercules, CA, USA).

The cells were lysed with CelLytic M solution (Sigma-Aldrich). After centrifugation, the supernatant was collected, mixed with SDS sample buffer, and boiled for 5 min at 98 °C. The sample was applied to SuperSep Ace gel (Wako), separated by a standard SDS-PAGE method, and then transferred onto polyvinylidene fluoride membranes, blocked with 5% skim milk and incubated at 4 °C overnight with anti-Akt, anti-p-Akt (S473) anti-p38, anti-p-p38 (T180/Y182), anti-ERK, anti-p-ERK (T202/Y204), anti-JNK, anti-p-JNK (T183/Y185) (dilution 1:3,000; Cell Signaling Technology), anti-cathepsin K (dilution 1:1,000; Santa Cruz), anti-coronin1A, anti-rab7 (dilution 1:3,000; Abcam), anti-LC3 (HRP conjugate) (dilution 1:2,000; MBL), anti-rab27a (dilution 1:3,000; R&D) and anti-β-actin (HRP conjugate) (dilution 1:2,000; Cell Signaling Technology). The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies. Chemiluminescence was detected using ImmunoStar LD (Wako), and images were captured by using LAS-4000 mini (Fujifilm) or Amersham Imager 600 (GE Healthcare).

Soluble recombinant mouse RANKL (sRANKL) was purchased from Peprotech (Rocky Hill, NJ, USA). The primary antibodies used were as follows: anti-TRAP, anti-cathepsin K, anti-c-Fos, anti-NFATc1, anti-Blimp-1, and anti-β-actin from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-carbonic anhydrase II, anti-cathepsin K, anti-HDAC6, and anti-IRF-8 from Abcam (Cambridge, MA, USA); and anti-Calcineurin from by MyBiosource (San Diego, CA, USA). Calcineurin inhibitors FK506 and cyclosporin A were purchased from Sigma-Aldrich (Saint Louis, MO, USA) and Abcam (Cambridge, MA, USA), respectively; the inhibitors were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) and stored at −20 °C. HDAC6 inhibitor CKD-WID was kindly provided by the Chong Kun Dang Pharmaceutical Corp. (Seoul, Korea).

Protein lysates were prepared from osteoclast precursors undergoing differentiation and from mature osteoclasts as previously described [15 (link)]. Cell lysates (25 μg) were boiled in the presence of sodium dodecyl sulfate (SDS) sample buffer (0.5 M Tris–HCl, pH 6.8, 10% wt/vol SDS, 10% glycerol, 0.05% wt/vol bromophenol blue) for 5 minutes and subjected to electrophoresis on 4% to 20% SDS -PAGE (Bio-Rad Laboratories, Hercules, CA, USA). Proteins were transferred to nitrocellulose membranes using a semidry blotter (Bio-Rad) and incubated in blocking solution (5% nonfat dry milk in TBS containing 0.1% Tween-20) for 1 hour to reduce nonspecific binding. Membranes were washed extensively and immunoblots were performed using anti-p-ERK (1:1000), anti-ERK (1:1000), anti-p-P38 (1:1000), anti-P38 (1:1000), anti-NFACT1 (1:1000), anti-CATHEPSIN K (1:1000), anti-DC-STAMP (1:500), anti-GAPDH (1:2000), anti-MMP9 (1:1000), anti-TRAP (1:200), and anti-β-ACTIN (1:4000) antibodies followed by rabbit anti-goat or goat anti-mouse antibodies respectively, conjugated to horseradish peroxidase (1:2000) in 5% milk, Santa Cruz Biotechnology). Western blots were developed using an enhanced chemiluminescence detection assay following the manufacturer’s directions (Bio-Rad).

Asiatic acid (HPLC > 98%) was purchased from the TransMIT (Gießen, Germany) and the compound at powder status was thawed in stock solution with dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA). Alpha–Minimal Essential Medium (α-MEM) and fetal bovine serum (FBS) as well as Trizol reagent were ordered from Life Technologies (Sydney, Australia). Anti-phosphate-ERK1/2, ERK1/2, anti-IkB α, anti-NFATc1, anti-c-Fos, and anti-Cathepsin K were purchased from Santa Cruz Biotechnology (Dallas, CA, USA). The loading control, anti-β-actin was obtained from Cell Signaling Technology (Danvers, MA, USA). The expression and purification of Glutathione S-transferase (GST)-rRANKL (GST-rRANKL) recombinant protein were performed as described (Xu et al., 2000 (link)). Macrophage colony stimulating factor (M-CSF) and ELISA kits of TRAcP and CTX-1 were obtained from R & D company (Minnneapolis, MN, USA).