Anti ctsk

Total protein was extracted from BMMs and C3H10 cells and from the bones of mice used in the OVX experiment using a radioimmunoprecipitation assay lysis buffer (RIPA) (Sigma-Aldrich), and the protein concentration was measured using a bicinchoninic acid protein (BCA) assay kit (EMD Millipore, Bedford, MA, USA). Protein equivalents were separated using 10% SDS PAGE and transferred onto PVDF membranes (EMD Millipore). The membranes were blocked for an hour at room temperature with 5% skim milk in Tris-buffered saline with Tween 20. After incubating primary antibodies overnight at 4 °C, the membranes were nurtured with corresponding HRP conjugated secondary antibodies (Proteintech Group, Chicago, IL, USA.) The bands were visualized employing ECL Luminous Liquid (EMD Millipore). ImageJ software was used to compute the relative grey level of proteins (NIH). Anti-TRAIL was procured from Santa Cruz Bio (Santa Cruz, CA, USA). Anti-CTSK, TRAP, NFATc1, OPG, RUNX2, OCN, RANKL, COL1a, p-P65, P65, p-P38, P38, p-JNK, JNK, p-ERK, ERK, p-IKBα, IKBα, IKKα, IKKβ, and p-IKKα/β were all purchased from Cell Signaling Technology (Danvers, MA, USA).

C57BL/6 mice (female, 6–8 weeks old) were purchased from the Laboratory Animal Center of Soochow University. During the experimental period, the mice were housed in ventilated cages in a specific pathogen‐free facility at room temperature (22–24°C) and kept on a 12‐h light/dark cycle, with water ad libitum and standard chow. P7C3 was obtained from MedChemExpress (Monmouth Junction, NJ, USA) and stored in dimethyl sulfoxide (DMSO) at −20°C. Recombinant mouse macrophage‐colony stimulating factor (M‐CSF) and RANKL were purchased from R&D Systems (Minneapolis, MN, USA). Soluble, recombinant human M‐CSF, RANKL and tumor growth factor β (TGF‐β) were obtained from PeproTech (London, UK). Anti‐Akt, anti‐phospho‐Akt, anti‐ERK, anti‐phospho‐ERK, anti‐p38, anti‐phospho‐p38, anti‐JNK, anti‐phospho‐JNK, anti‐NFATc1, anti‐CTSK, anti‐MMP‐9, and anti‐GAPDH antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti‐TRAF‐6, anti‐IκB, anti‐p65‐NF‐κB, and anti‐phospho‐p65‐NF‐κB antibodies were purchased from Abcam (Cambridge, MA, USA). Alpha‐minimum essential medium (α‐MEM), fetal bovine serum (FBS), streptomycin, and penicillin were obtained from Gibco BRL (Grand Island, NY, USA).

The cells were first washed three times with PBS and lysed with radioimmunoprecipitation assay buffer with complete protease inhibitor (Solarbio). After protein quantification, the samples were separated by 12% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Bio-Rad) according to the standard protocol. The membranes were blocked with 5% skim milk and incubated overnight at 4 °C with the appropriate primary antibodies. The primary antibodies were as follows: anti-TRAP, anti-CTSK, anti-NFATc1, and anti-Itgb1 (1:1000; Cell Signaling Technology). β-Actin was obtained from Affinity Biosciences. The membranes were subsequently incubated with secondary antibodies for 1 h at RT and washed with a Tris-buffered saline with Tween-20 for 30 min. The blots were visualized using Tanon image scanning. ImageJ software was used for quantitative analysis of the blots.