Primary antibody against gapdh

Total protein from cell lines was extracted using a whole-cell extraction buffer. Protein samples were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore Corporation, Bedford, MA, USA). After blocking, the membranes were incubated overnight at 4°C with the following primary antibodies: HtrA2 (1 : 1000) and GAPDH (1 : 2500). Primary antibody against HtrA2 was purchased from ZEN BIO, China. The primary antibody against GAPDH was purchased from Abcam, Cambridge, UK. Subsequently, the membranes were developed with secondary peroxidase-coupled anti-rabbit antibodies, followed by chemiluminescence imaging. Band intensity was quantified using ImageJ software.

DEN (molecular formula: C₁₆H₂₅NO₂, MW: 263.19, and CAS No. 2115-91-5) was purchased from Chengdu Pufei De Biotechnology Co., Ltd. (Chengdu, China). Recombinant human IL-1β was purchased from PeproTech (Cranbury, NJ, USA). CCK-8 was obtained from Dojindo (Kumamoto, Japan). The cell culture reagents were purchased from Gibco (Grand Island, NY, USA). The primary antibody against GAPDH was obtained from Abcam (Cambridge, MA, USA). The primary antibodies against Col2a1, p21, and ACAN were obtained from Abcolonal (Wuhan, China). The primary antibodies against TNF-α, MMP13, ADAMTS5, p65, β-Actin, and p16^INK4a were purchased from Proteintech (Wuhan, China). The primary antibodies against IL-6, IκB-α, and Lamin B were purchased from Abmart (Shanghai, China).

Liver tissues or cells were lysed in RIPA buffer with complete protease inhibitor (Sigma). Nucleoprotein was prepared as previously described (Ho et al., 2010 (link)). Then whole lysates (30 μg) were separated by polyacrylamide gel electrophoresis and proteins were transferred to polyvinylidene difluoride (PVDF) membranes. These membranes were incubated with primary antibodies against BUB3 (1:2,000, CY8528), CHK1 (1:1,000, CY5063), cyclin A (1:1,000, CY1026), MCM5 (1:2,000, CY7185), P21 (1:1,000, 27296-1-AP), Ube2c (1:1,000, 12134-2-AP), and GAPDH (1:2,000, ab9485) overnight at 4°C followed by incubate with secondary antibodies [goat anti-rabbit secondary antibody (HRP)] 2 h at room temperature. Immune complexes were detected using the Enhanced Chemiluminescence System (Thermo Fisher Scientific) and the intensity of each band was quantified using ImageJ software. Primary antibodies against Bub3, CHK1, cyclin A, and MCM5 were purchased from Abways (Shanghai, China). Primary antibodies against P21 and Ube2c were purchased from Proteintech (Wuhan, Hubei, China). Primary antibody against GAPDH was purchased from Abcam (Shanghai, China).