Anti trim33

HOB and MC3 T3‐E1 cells were cultured on a coverslip. When cell confluence reached 95%–100%, the cells were washed with PBS thrice (10 min each time). After fixation in 4% formaldehyde (Aladdin) for nearly 20 min at room temperature, the cells were washed with PBS again. Then, the cells were permeabilized with 0.4% Triton X‐100 (Thermo Fisher) for nearly 1 h and blocked in BSA buffer for about 1 h at room temperature. Following this, the cells were incubated with the primary antibodies anti‐FOXO3a (Abcam, 5 µg/ml) and anti‐TRIM33 (Abcam, 1 µg/ml) for nearly 24 h at 4 °C and then incubated with FITC‐conjugated secondary antibody for about 1 h in the dark. The cells were then washed with PBS and stained with DAPI. The coverslips were sealed with glycerin and observed under a fluorescence microscope (Olympus, Japan).

The cell line used in this study was purchased from the Chinese Academy of Sciences cell bank. The antibodies being used included rabbit anti-TRIM33 (Abcam, 47062 and 84455), anti-β-actin (Abcam, 8227), anti-E-cadherin (Abcam, 15148), anti-N-cadherin (Abcam, 18203), anti-vimentin (Abcam, 92547), anti-β-catenin (Abcam, 16051), anti-cyclin D1 (Abcam, ab40754), and anti-c-myc (Abcam, 32072) antibodies. Wnt agonist 1 was purchased from Selleck Chemicals (Shanghai, China) and was used at a final concentration of 10 μM at 37°C for 24 h.

Total proteins were extracted from HOB and MC3 T3‐E1 cells, the primary osteoblasts isolated from the bone tissues of patients with osteoporosis and osteoarthritis or the osteoblasts isolated from the mouse bone marrow. The protein concentrations were tested using the BCA Protein Assay Kit (Beyotime). In total, 30 μg of protein from each sample was applied for SDS‐PAGE. After separation, the proteins were transferred into PVDF membranes (Roche, Basel, Switzerland). The PVDF membranes were blocked for nearly 1 h and then incubated with the primary antibodies anti‐FOXO3a (Abcam, 1/2500), anti‐TRIM33 (Abcam, 1/1000), anti‐Lamin B (Abcam, 1/2000), anti‐GAPDH (Abcam, 1/2500), anti‐CBP (Abcam, 1/1000), anti‐Gadd45a (Abcam, 1/1000), anti‐FOXO1 (Abcam, 1/2000), anti‐FOXO4 (Abcam, 1/2000) and anti‐catalase (Abcam, 1/2000) at 4°C for about 24 h. This was followed by the incubation with the corresponding secondary antibodies (Abcam, 1/2000) at room temperature for nearly 1 h. Protein bands were visualized using BeyoECL Moon (Beyotime). LaminB and GAPDH were applied as the references for nuclear protein and total protein, respectively.