Ripa buffer

Total cell extracts were prepared in RIPA buffer (RiboBio, Guangzhou, China) supplemented with a complete protease inhibitor cocktail (Invitrogen, Carlsbad, CA, USA) at 4°C for an hour. The lysates were cleared by centrifugation at 12000 rpm for 15 min, and the protein concentrations of the samples were determined using a Pierce™ BCA protein assay kit (Thermo Fisher Scientific, Pittsburgh, PA, USA). The lysates were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membrane filters, and probed with anti-TRIM11 polyclonal antibody and anti-β-actin mouse antibody (Sigma-Aldrich, St Louis, MO, USA), following by incubating with horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotech., Santa Cruz, CA, USA). Densitometry of obtained signals was semi-quantified with the ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Osteoblasts were lysed with 1 ml RIPA buffer (Guangzhou RiboBio Co., Ltd.) to extract total protein. Protein samples were quantified using a bicinchoninic acid kit (Sangon Biotech Co., Ltd.), followed by denaturation in boiling water for 5 min. Electrophoresis was performed using 12% SDS-PAGE to separate proteins (30 µg per well) according to their molecular weights. Proteins were transferred to a PVDF membrane and blocking was carried out in 5% non-fat milk for 2 h at room temperature. Primary antibodies of rabbit GAPDH (1:1,200; cat. no. ab181602; Abcam) and p53 (1:1,200; cat. no. ab131442; Abcam) were used to incubate the membranes for 15 h at 4˚C. Horseradish peroxidase goat anti-rabbit (immunoglobulin G; 1:1,100; cat. no. ab6721; Abcam) secondary antibody was then used to blot the membranes further at room temperature for 2 h. The ECL Chemiluminescence Detection kit (Sangon Biotech Co., Ltd.) was used to develop protein signals. Gray values were normalized using ImageJ version 1.46 (National Institutes of Health).