Beyogel plus page

A Native-PAGE was applied to analyze the molecular structures of the untreated and sonicated OVM. The precast BeyoGel™Plus PAGE (Beyotime Biotechnology Co. Ltd, Shanghai, China) included 4 % stacking gels and 15 % separating gels. 80 μL of sample was mixed with 20 μL loading buffer (4 × ). Gel board was mounted into a Biorad Mini-PROTEAN Tetra Electrophoresis System (Biorad, California, US), and 20 μL of each sample mixture was loaded into gel lanes. The running buffer was prepared by Tris-Gly Native-PAGE buffer powder. A color mixed protein marker of 11–180 kDa (Solarbio, Beijing, China) was utilized to estimate the molecular mass of each sample. The whole electrophoresis process kept the operating voltage at 150 mV.

Total protein was separated by 4-20% Tris-HCl/SDS-polyacrylamide gels (Beyotime, BeyoGel™ Plus PAGE, China) and transferred to PVDF membranes. Then, the membranes were incubated with blocking buffer (Beyotime, QuickBlock™ Western, China) for 1h, with primary antibodies (IGFBP5 (1:1000, A12451, ABclonal), β-actin (1:5000, ab8226, Abcam)) overnight at 4 °C and with secondary antibody (1:5000, HRP Donkey Anti-Rabbit IgG (H+L) (AS038), ABclonal) for 1h at room temperature. Membranes were imaged by ChemiDoc™ Touch (ChemiDoc Touch 1708370, Bio-red, USA).

The total protein of primary osteoblasts was extracted using RIPA lysis buffer (Beyotime, China) and quantified by BCA assay (Beyotime, China). Equal amounts of proteins (100 μg) were separated via BeyoGel™ Plus PAGE (Beyotime, China) and then transferred to a PVDF membrane (Millipore, USA). After transferring, the membranes were blocked with 5% fat-free milk for 1 h. The membranes were incubated with primary antibodies (Bax, ab32503; Caspase-3, ab32351; and cleaved-Caspase-3, ab32042) purchased from Abcam (USA) and GAPDH, 10494-I-AP purchased from Proteintech (China) at 4°C overnight. The membranes were incubated with second antibodies (goat anti rabbit IgG HRP SE134, goat anti mouse IgG HRP SE131, Solarbio, China) at 37°C for 1 h. The ECL system (CLINX, China) was used for exposing protein bands. The intensity of the bands was analyzed using Image Lab (version 3.0, Bio-Rad, USA).