Proteasome inhibitor

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).

Protein extracts from cells were prepared in RIPA lysis buffer supplemented with a proteasome inhibitor (Beyotime, Haimen, China). Total proteins were separated by using 10% SDS-PAGE and then transferred to a PVDF 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 (RC-5G5; 1:10000; KangChen Bio-tech, Shanghai, China), TRAF6 (ab33915; 1:8000; Abcam, Cambridge, UK), p-p38 MAPK (#4511; 1:1000; CST, Boston, USA), STE20 (ab155198; 1:3000; Abcam, Cambridge, UK), and IGF1R (ab79176; 1:2000; Abcam, Cambridge, UK). HRP goat anti-rabbit IgG (BA1054; 1:20000, Boster Biologic Technology, Wuhan, China) was used as a secondary antibody for 2 h at room temperature. The signal intensity was quantified using Image J software (National Institute of Health, Bethesda, Maryland, USA).

Protein extracts of BMSCs were prepared with RIPA lysis buffer containing proteasome inhibitor (Beyotime). The total protein was separated using 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. After 1 h of blockade with 5% bovine serum albumin, membranes were incubated with antibodies OPN (ab283656, 1 : 1000), OCN (ab93876, 1 : 100), NEDD4L (ab245522, 1 : 2000), HuR (ab200342, 1 : 1000), FOXA2 (ab256493, 1 : 1000), and β-actin (ab8227, 1 : 1000) at 4°C overnight and with the secondary antibody (ab205718, 1 : 2000) for 1 h. Immunoreactive bands were observed using the enhanced chemiluminescence assay reagent (Millipore), and the grayscale value was quantified using a chemiluminescence determination system (Bio-Rad, Hercules, CA, USA). All antibodies were provided by Abcam.

To determine the protein expression of certain markers, protein extracts from BMSCs (day 3 or day 7 after the induction of osteogenesis) were prepared in radioimmunoprecipitation assay lysis buffer (Beyotime) supplemented with a proteasome inhibitor (Beyotime). Total proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride membrane (Millipore, Shanghai, China). The membranes were then blocked in 5% BSA at room temperature for 1 h and then incubated overnight at 4 °C with antibodies specific to GAPDH (1:2000, CST), RUNX2 (1:1000; CST), COL1A1 (1:1000; Abcam), t-AKT (1:1000; CST), p-AKT (1:1000; CST), t-p38 (1:1000; CST), p-p38 (1:1000; CST), or β-catenin (1:1000; CST). After that, a secondary antibody (1:5000, Boster Biologic Technology, Wuhan, China) was applied for 2 h at room temperature. The immunoreactive bands were finally visualized and quantified using a Bio-Rad XRS chemiluminescence detection system (Bio-Rad, CA, USA).

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).