Anti sapk jnk

Whole-cell lysates were prepared, and proteins were extracted (n = 3 independent experiments). The following antibodies were used for incubation: anti-ADAMTS-4/5, anti-type X collagen and anti-MMP13 (Abcam), anti-CaM kinase II (CaMKII) (pan), anti-phospho-CaMKII (Thr286), anti-phospho-stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) (Thr183/Tyr185) (81E11), anti-SAPK/JNK, anti-nuclear matrix protein p84 (Abcam), and anti-phospho-protein kinase C (PKC) (pan) (βII Ser660) were purchased from Cell Signaling Technology (Ozyme, Montigny-le-Bretonneux, France). Samples were treated and analyzed with the Active Rho Pull-Down and Detection kit (Pierce Biotechnology, Rockford, IL, USA).

After removing the supernatant, the cells were washed twice with PBS at 4°C. RIPA lysis buffer (Beyotime, China) containing 1% protease inhibitor (Sigma–Aldrich, USA) and 1% serine protease inhibitor (Sigma–Aldrich, USA) was added for 30 min to lyse cells and extract proteins from each sample. The concentrations of total proteins were detected by a BCA protein assay kit (Beyotime, China) according to the manufacturer’s instructions. Protein samples were mixed with 5× loading buffer (ThermoFisher, USA) at a ratio of 4:1 and boiled at 99°C for 10 min. Samples were loaded and run on SDS–PAGE gels (CWBIO, China) and transferred onto PVDF membranes (Millipore, USA). Membranes were blocked with 2% skim milk (BD, USA) for 1 h at room temperature and then incubated with anti-IKKα, anti-IKKβ, anti-pIKKα/β, anti-p65, anti-pp65, anti-p38, anti-pp38, anti-SAPK/JNK, anti-pSAPK/JNK, and anti-GAPDH primary antibodies (Abcam, UK) overnight at 4°C. After primary incubation, blots were washed and incubated with secondary goat anti-rabbit or goat anti-mouse HRP (Abcam, UK) for 1 hour. Membranes were washed and exposed to chemiluminescent HRP substrate (Millipore, USA). Images were obtained using the GeneGnome XRQ system (Syngene, USA) and analyzed using ImageJ software.