Ab252944

Total protein was extracted from cells and then lysed using radio-immunoprecipitation assay lysis buffer (Boster Biological Technology Co., Ltd., Wuhan, Hubei, China) containing protease inhibitor on ice for 30 min. A bicinchoninic acid assay kit (Boster) was employed to determine the protein concentration. The protein was subsequently separated using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The membrane was blocked with 5% skimmed milk at indoor temperature for 1 h to block nonspecific binding and probed overnight at 4 °C with diluted primary antibodies against HDAC1 (ab109411, 1:1000, Abcam Inc., Cambridge, UK), Snail (ab216374, 1:1000, Abcam), TPX2 (ab252944, 1:1000; Abcam). After washing, the membrane was re-probed at indoor temperature with horseradish peroxidase-labeled secondary goat anti-rabbit immunoglobulin G (IgG) (ab205719, 1: 2000, Abcam Inc., Cambridge, UK) for 1 h. The immunocomplexes on the membrane were visualized using enhanced chemiluminescence reagent (EMD Millipore, Billerica, MA) and band intensities were quantified using the ImageJ software with β-actin serving as a loading control.

Protease and phosphatase inhibitors were added to RIPA buffer (Beyotime Biotechnology, Jiangsu, China), which was used to lyse the cells. A BCA protein assay kit from Thermo Fisher Scientific (Waltham, MA, USA) was applied for protein content measurement. SDS-PAGE was used to separate equal quantities of protein, which was then deposited onto PVDF membranes from Millipore. After blocking with 5% non-fat milk, the membranes were incubated overnight at 4 °C with primary antibodies against TPX2 (ab252944; Abcam, Cambridge, UK), MMP13 (ab315267; Abcam, Cambridge, UK), cleaved caspase-3 (ab32042; Abcam, Cambridge, UK), or GAPDH (ab8245; Abcam, Cambridge, UK), all diluted at a 1:1,000 concentration. The protein bands were detected using Thermo Fisher Scientific’s ECL substrate after being incubated with an HRP-conjugated secondary antibody, and their quantities were calculated using ImageJ software.

Immunofluorescence staining was conducted to investigate the distribution of TPX2 (ab252944; Abcam, Cambridge, UK) and MMP13 (ab315267; Abcam, Cambridge, UK) in the LPS-treated human normal chondrocytes. The cells were permeabilized with 0.1% Triton X-100, fixed by 4% paraformaldehyde, and blocked with 5% BSA. TPX2 and MMP13-specific primary antibodies were then used to incubate the cells, followed by the proper secondary antibodies. DAPI was used to stain the nuclei. With the use of a fluorescent microscope, pictures were taken.