Efficient chemiluminescence ecl kit

Total protein was isolated from cells using Protein Lysis Buffer (Beyotime, Shanghai, China). Equivalent amounts of proteins (25 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, the proteins were transferred to the polyvinylidene fluoride (PVDF) membranes. After blocking with 3% skim milk, the membranes were incubated with primary antibodies at 4°C overnight. Next, the membranes were incubated with secondary antibody (Abcam; 1:5000) for 1 h at room temperature. The efficient chemiluminescence (ECL) kit (Thermo Fisher Scientific) was used to detect the protein bands. The primary antibodies used in the present study were as follows: anti-PDHX (1:1000), HIF-1α (1: 1000), anti-β-actin (1:1000). The β-actin worked as an internal control.

The total protein extraction kit (KeyGen BioTECH, Jiangsu, China) was used to isolate proteins from the above 10 patients. The protein lysates were separated by 10% sodium dodecyl sulphate—polyacrylamide gels (SDS-PAGE) (Beyotime, Shanghai, China), and then transferred onto polyvinylidene difluoride (PVDF) membranes (Beyotime, Shanghai, China). Primarry antibodies against RAF1, phospho-RAF1 (p-RAF1) and GAPDH were purchased from Abcam (ab-137435, ab-60985, ab-181602; Abcam, Cambridge, UK) and were diluted at 1:1000, 1:1000 and 1:3000. The secondary antibody goat anti-rabbit IgG (Beyotime, Shanghai, China) was diluted at 1:5000. The Efficient chemiluminescence (ECL) kit (Thermo, Shanghai, China) was utilized to detect horseradish peroxidase (HRP) and its products.

According to the instructions of ProteoPrep® Total Extraction Sample Kit (Thermo Fisher Scientific, Waltham, MA, USA), the total protein in cells and cartilages of rats was extracted. The protein concentration was determined by BCA Kit (Wuhan Boster Biotechnology Co., Ltd., Wuhan, China). Protein samples (30 μg) were separated by 10% sodium dodecyl sulfate-Polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred into polyvinylidene fluoride (PVDF) membrane (Thermo Fisher Scientific). The membranes were incubated with blocking buffer (5% skim milk) for 2 h at room temperature and then incubated with the primary antibody (p-p65, 3033; p65, 8242; IκBα, 4814; p-IκBα, 2859; GAPDH, 5174; 1 : 1000, Cell Signaling Technology, USA; GAPDH as internal control) overnight at 4°C. Subsequently, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit (1: 4000; Shanghai MiaoTong Biotechnology Co., Ltd, Shanghai, China) for 2 h. Protein bands were visualized according to the instructions of efficient chemiluminescence (ECL) kit (Thermo Fisher Scientific) and analyzed with the Image LabTM Software (Bio-Rad, USA).