Ecl reagent

The total proteins of MC3T3-E1 cells or BMSCs were extracted and lysed with RIPA buffer (Beyotime, China), and protein concentration was detected using a BCA protein assay kit (Solarbio, China). Then, a 20 μg total protein sample was loaded onto 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for separation and transferred to a PVDF membrane. After being blocked with 5% skimmed milk, the membrane was incubated at 37°C with the primary antibodies against caspase-3 (ab13847, Abcam), caspase-9 (ab32539, Abcam), SIRT1 (ab110304, Abcam), BMP-2 (ab214821, Abcam), RANKL (ab45039, Abcam), OPG (ab73400, Abcam), IκBα (ab32518, Abcam), p-IκBα (AF2002, Affinity), p65 (ab207297, Abcam), p-p65 (AF2006, Affinity), IKKα (ab32041, Abcam), p-IKKα (AF3013, Affinity), NFATc1 (ab2796, Abcam), and cathepsin K (ab19027, Abcam) at 4°C overnight. After that, the membrane was further incubated with the secondary antibody at room temperature for 1 h. Then, the enhanced chemiluminescence (ECL) reagent (Solarbio, China) was used to visualize the protein bands, and the relative band density was semiquantified with the ImageJ software.

The total protein was extracted with a RIPA lysis buffer, and its concentration was determined with a BCA kit (Solarbio). The polyacrylamide gel was prepared in advance, and its concentration was determined by the size of the protein to be detected. The protein sample was loaded into a polyacrylamide gel, followed by electrophoresis for 2–3 h. Afterward, the protein was transferred onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA), and skim milk was used to block the heterogenetic antigens. Subsequently, the membrane loaded with protein was incubated with primary antibody at 4°C in the dark overnight. After rinsing with TBST buffer, the membrane was incubated with a secondary antibody labeled with HRP, and reacted with ECL reagent (Solarbio) for several minutes, followed by signal exposure in the dark. The antibody information was shown in the following: RASIP1 (1:1000; Affinity, Changzhou, Jiangsu, China), cyclin E1 (1:1000; Affinity), cyclin B1 (1:1000; Affinity), cyclin D1 (1:1000; Affinity), p27 (1:1000; Affinity), matrix metalloproteinase 2 (MMP2) (1:2000; Novus Biologicals, Littleton, CO, USA), MMP9 (1:1000; Affinity), cleaved caspase-3 (1:1000; Affinity), Bax (1:1000; Affinity), Bad (1:1000; Affinity), Bcl-xl (1:1000; Affinity), FOXO3 (1:500; Affinity), GAPDH (1:10000; Affinity).

After extraction of total protein by using RIPA lysate (Solarbio, Beijing, China), the BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) was utilized to measure the protein concentrations. Equal amounts of proteins (20 μg) were separated on a 10% SDS/PAGE gel and electrotransferred (100 V, 2 h) to nitrocellulose membranes. The membranes were blocked with 5% BSA for 1 h and were then incubated with primary antibody for LETM2 (Proteintech, Wuhan, China) and GAPDH (Abcam, Cambridge, UK) overnight at 4 °C. The corresponding secondary antibodies were applied on the following day. The blots containing target bands were exposed by enhanced chemiluminescence (ECL) reagent (Solarbio, China) on the Exposure meter. The densitometry readings of each band were detected by ImageJ software 1.8.0 in the gray value analysis, and the relative expression was calculated as intensity ratio = target protein gray density/GAPDH gray density

Whole-cell lysates were extracted using RIPA Cell Lysis Buffer (Solarbio, Beijing, China) containing a protease inhibitor mixture (Solarbio, China). The concentration of protein in the extract from MAC-T cells was determined using a BCA Protein Assay Kit (Solarbio, China). Extracts containing equal quantities of proteins (50 μg) were resolved on 12% SDS-polyacrylamide gel and transferred to PVDF membrane and blocked with 5% non-fat dry milk in TBST for 2 h. The membrane was then incubated with primary antibody overnight at 4 °C. Primary antibodies were purchased from Beyotime Institute of Biotechnology (Haimen, China) at a 1:1000 dilution. After washing with TBST, the members were incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. Finally, specific bands were produced using ECL reagent (Solarbio, China) and digitally detected with the GelDocTM XR Plus system (Bio-Rad, USA).

Cells were lysed in ice-cold RIPA lysis buffer (R0020, Solarbio), and protein levels were quantified using a BCA protein assay kit (P0012S, Beyotime). The extracted cellular proteins were mixed with loading buffer (v/v = 4:1) (P1040, Solarbio) and boiled for 10 min. Protein samples (30 μg per well) were separated by 8% SDS–PAGE gels and transferred to PVDF membranes. The membranes were blocked at room temperature with 5% BSA in 1 × TBST (0.2% Tween-20, pH = 7.6) buffer for 2 h and then incubated with primary antibodies against GAPDH (1:100,000, A19056, ABclonal, Wuhan, China) and ARID5B (1:2000, NBP1-83622, Novus, Shanghai, China) at 4 °C overnight. After being washed with 1 × TBST buffer four times (10 min each time), the membranes were then incubated with HRP-conjugated mouse anti-rabbit (1:5000, AS061, ABclonal) and goat anti-mouse (1:5000, AS003, ABclonal) secondary antibodies at room temperature for 1 h. Protein bands were visualized by chemiluminescence using an ECL reagent (PE0010, Solarbio) and photographed by a Tanon-5200 Chemiluminescent Imaging System (Tanon, Shanghai, China). Image-Pro Plus (v 6.0) software was used to perform the densitometric semiquantitative analysis of protein bands. The protein expression was normalized against GAPDH.