Enhanced chemiluminescent detection reagent

To determine the protein expression of osteoclast‐related markers, BMMs (1.2 × 10⁵/well) were seeded into 6‐well plates and cultured with 0 or 1000 ng/mL IGFBP7 for 0, 1 and 3 days. To discover the underlying signalling pathways, RAW264.7 cells (5 × 10⁵ cells per well) were seeded into 6‐well plates. RAW264.7 cells were pre‐treated with vehicle or IGFBP7 (1000 ng/mL) for 6 h and thereafter exposed to RANKL (100 ng/mL) for the indicated time (0, 5, 10, 20, 30 and 60 min). Total cellular protein extracts were obtained using RIPA lysis buffer with a protease inhibitor and a phosphatase inhibitor (Beyotime). Equal amounts of protein extracts were separated by 10% SDS‐PAGE and then electroblotted to PVDF membranes (Millipore). The membranes were thereafter blocked with 5% BSA for 1 hour and then incubated with primary antibodies at 4°C overnight. After that, the membranes were probed with secondary antibodies (BOSTER) for 2 hours at 4°C. The immunoreactive bands were detected by an enhanced chemiluminescent detection reagent (Millipore) in a Bio‐Rad XRS chemiluminescence detection system (Bio‐Rad).

Cells were lysed in RIPA lysis buffer (Beyotime) with a proteasome inhibitor (Beyotime). Total proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane (Millipore, Shanghai, China). After blocking in 5% nonfat milk for 2 h, the membranes were incubated overnight at 4°C with antibodies specific for glyceraldehyde 3-phosphate dehydrogenase (A00227-1; 1:8,000; Boster Biological Technology, Wuhan, China), RUNX2 (#12556S; 1:1,000; Cell Signaling Technology), PPARγ (#2435S; 1:1,000; Cell Signaling Technology), or SIRT1(#8469S; 1:1,000; Cell Signaling Technology). Horseradish-peroxidase-conjugated goat anti-rabbit IgG (BA1056; 1:5000; Boster Biological Technology) was used as a secondary antibody for 2 h at room temperature. The immunoreactive bands were detected using an enhanced chemiluminescent detection reagent (Millipore, Shanghai, China). Signal intensity was measured using a Bio-Rad XRS chemiluminescence detection system (Bio-Rad, Hercules, CA, USA).

The middle lobe of the liver was placed in lysis buffer (containing protease and phosphatase inhibitor) and homogenized by using an ultrasonic cell crusher (scientz-II D, Ningbo Scientz Biotech Co., Ltd., Ningbo, China). Then, the targeted proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then blotted onto a nitrocellulose (NC) membrane. Thereafter, the NC membrane was incubated with the corresponding primary and secondary antibodies. After incubation, proteins were examined by using a VILBER Fusion FX7 imaging system (Vilber Lourmat, Marne-la-Vallée, France) with an enhanced chemiluminescent detection reagent (Millipore, Billerica, MA, USA).

Proteins were extracted from cells by lysing in RIPA buffer supplemented with protease inhibitors and phosphatase inhibitors. After protein quantification, cell lysates were suspended in 5x loading buffer and then equal amounts of proteins were subjected to migration on 4%–20% polyacrylamide linear gradient gels, then transferred to a polyvinylidene fluoride membrane (Millipore). The membranes were blocked by nonfat milk (5%) for 60 min and then incubated with primary antibodies overnight at 4°C. Horseradish peroxidase-conjugated goat antirabbit IgG (1 : 5,000, Beyotime) was used as a secondary antibody for 1 hr at room temperature. Immunoreactive bands were detected using an enhanced chemiluminescent detection reagent (Millipore). The signal intensity was measured using a Bio-Rad XRS chemiluminescence detection system (Bio-Rad, Hercules, CA, United States).

Cells were lysed in RIPA lysis buffer supplemented with a proteasome inhibitor (Beyotime). Total proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane (Millipore, Shanghai, China). After blocking in 5% non-fat milk for 2 h, the membranes were incubated overnight at 4 °C with antibodies specific to β-actin (1:1000, Abcam Inc., Waltham, MA, USA) or β-catenin (1:1000, Cell Signaling Technology, Danvers, MA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:1500; Cell Signaling Technology, USA) was applied as a secondary antibody for 2 h at room temperature. The immunoreactive bands were detected using an enhanced chemiluminescent detection reagent (Millipore, Shanghai, China). Signal intensity was measured using a Bio-Rad XRS chemiluminescence detection system (Bio-Rad, Hercules, CA, USA).

Protein extracts from cells were prepared in RIPA lysis buffer supplemented with a proteasome inhibitor (Beyotime, Haimen, China). Total proteins were separated by 10% SDS-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride membrane (Millipore, Shanghai, China). After blocking in 5% BSA for 1 h at room temperature, the membranes were incubated overnight at 4 °C with antibodies specific to GAPDH (1:2000, Cell Signalling Technology), FOXA2 (1:1000; Cell Signalling Technology), RUNX2 (1:1000; Cell Signalling), COL1A1 (1:1000; Abcam), t-ERK (1:1000; Cell Signalling Technology) or p-ERK (1:2000; Cell Signalling Technology). Horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000, Boster Biologic Technology, Wuhan, China) was used as a secondary antibody for 2 h at room temperature. The immunoreactive bands were visualised using an enhanced chemiluminescent detection reagent (Millipore). Signal intensity was quantified using a Bio-Rad XRS chemiluminescence detection system (Bio-Rad, Hercules, CA, USA).

Cells were lysed and the total cell protein was extracted in RIPA buffer containing a proteasome inhibitor (Beyotime, China). Protein concentrations were measured using a BCA protein assay kit (Beyotime). Equal protein amounts were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Shanghai, China). After blocking in 5% defatted milk for 2 h, the membranes were incubated with specific primary antibodies overnight at 4°C. Primary antibodies used in the present study include anti-GAPDH (1:10000, Proteintech, United States), anti-POSTN (1 μg/ml; Abcam, China), anti-SOST (1 μg/ml; Abcam), anti- RUNX2 (1 μg/ml; Abcam), anti-COL1A1 (1: 1000; Abcam), anti-Active (non-phosphorylated) β-catenin (1: 1000; Cell Signaling Technology), and anti-Total β-catenin (1: 1000; Cell Signaling Technology). After washing with TBST for four times, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Sigma, United States) for 1 h at room temperature. After washing with TBST for five times, the PVDF membranes were analyzed using an enhanced chemiluminescent detection reagent (Millipore) and scanned with the BioSpectrum Imaging System (UVP, Germany).