Horseradish peroxidase labelled secondary antibody

TRIzol, Lipofectamin™ 2000 transfection Kit (#11668027), and Power SYBR Green PCR Kit (#4309155) were purchased from Invitrogen, USA. Dual-Luciferase Reporter Assay System and M-MLV reverse transcriptase were obtained from Promega. The mirVana™ qRT-PCR miRNA Detection Kit (#AM1558) was acquired from Life Technologies, USA. The miR-24 mimics and inhibitors were synthesised by the GenePharma Company (Shanghai, China). MTT Detection Kit and TUNEL Apoptosis Detection Kit were procured from Beyotime Institute of Biotechnology (Beijing, China). Lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) detection kits were procured from the Nanjing Jiancheng Bioengineering Institute, China. Annexin V Apoptosis Detection Kit was purchased from BD Biosciences, USA. Bim and GAPDH antibodies were purchased from Cell Signaling. The horseradish peroxidase-labelled secondary antibody was procured from Santa Cruz Biotechnology. The enzyme label instrument, Tecan Infinite M1000, was procured from Switzerland. The ABI 7300 real-time PCR instrument was purchased from Applied Biosystems, USA. The NanoDrop 2000 spectrophotometer was purchased from Thermo, USA. The FacsCalibur flow cytometer was purchased from BD Biosciences, USA. The inverted fluorescence microscope was purchased from Olympus, Japan. The upfront fluorescence microscope was purchased from Leica, Germany.

Western blot assay was used to detect protein expression levels. First, cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (25 mmol/L Tris∙HCl pH7.6, 150 mmol/L NaCl, 1% NP‐40, 0.25% sodium deoxycholate, 0.1% SDS) with protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Then, the protein lysates were denatured at 95°C for 5 minutes after mixing with 5x SDS‐loading buffer. Subsequently, the cell extracts (30 µg protein) were separated on a sodium dodecyl sulphate‐polyacrylamide electrophoretic gel (SDS‐PAGE) and then transferred to nitrocellulose membranes. After blocked with 3% BSA for 2 hours, the membranes were incubated overnight at 4°C with the following primary antibodies at dilutions of 1:1000: PARP (#9532), Bcl‐2 (#2876), Bax (#14796), Caspase‐9 (#9502), Caspase‐3 (#9664), CyclinD1 (#2978), CDK2 (#2546), CDK4 (#12790), CDK6 (#13331), CyclinD1 (#2978) purchased from Cell Signaling Technology, and PCNA (#ab29) obtained from Abcam. Next, the membrane was incubated with the corresponding horseradish peroxidase–labelled secondary antibody (Santa Cruz Biotechnology) for 2 hours at room temperature. Lastly, the signal was visualized by an enhanced chemiluminescence (ECL) kit (Immobilon Western HRP, MILLIPORE, USA) according to the manufacturer's instructions.

Total protein samples were extracted using a radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology, Nantong, China) containing 1% protease inhibitor (Cell Signaling Technology). A BCA Protein Assay Kit (Beyotime Biotechnology, Nantong, China) was used to detect protein concentrations. Total protein samples (20 μg) were separated using 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) and subsequently transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with primary antibodies specific for anti‐FOXP1 (ab134055, 1:1000), anti‐OCN (ab93876, 1:500), anti‐RUNX2 (ab23981, 1 μg/mL) and anti‐GAPDH (ab9485, 1:2500) (Abcam, Cambridge, UK, USA) at 4°C overnight. They were then incubated with horseradish peroxidase‐labelled secondary antibodies (Santa Cruz, Dallas, TX, USA) for 2 hours at room temperature. An enhanced chemiluminescence (ECL) kit (Solarbio, China) was used to visualize protein bands.