Anti pi3k antibody

Protein expressions were semi-quantitatively analyzed by western blot. Radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) was added to cell cultures, and proteins were collected to be prepared for gel electrophoresis. The loading buffer and the reagents for preparing gels were purchased from Beyotime. Then, proteins were isolated by gel electrophoresis and transferred to the polyvinylidene fluoride (PVDF) membranes, which were blocked by being incubated with 5% BSA (Sigma-Aldrich) or 5% nonfat-dried milk/tris-buffered saline with Tween 20 (TBST) for 1 hour at room temperature. Successively, PVDF membranes were gently shaken with the primary antibodies at 4 ℃ for more than 10 hours and with the secondary antibody for 1 hour at room temperature. The antibodies included anti-pan-Akt antibody, anti-phosphorylated Akt (S473) antibody (CST), anti-PI3K antibody, and anti-GAPDH antibody (Abcam). After washing the membranes 3 times with TBST in the interval of 2 incubations, the membranes were incubated with the chemiluminescence reagent (Merck Millipore, Billerica, MA, USA).

Chondrocytes were washed twice with phosphate-buffered saline (PBS) and lysed using radioimmunoprecipitation assay lysis buffer (CW Biotech, Beijing, China), and the protein concentrations were measured by a bicinchoninic acid protein assay kit (Thermo Fisher Scientific Inc., Waltham, USA). An equal amount of total protein was then separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes using β-actin as an endogenous control. The membranes were blocked with 5% skim milk for 2 h at room temperature and then incubated overnight at 4°C with primary antibodies, including anti-WISP1 antibody, anti-PI3K antibody, anti-p-PI3K antibody, anti-p65 antibody, anti-p-p65 antibody, anti-β-actin antibody (Abcam, Cambridge, UK). After washing three times with tris buffered saline with Tween-20, the membranes were incubated with the secondary antibody for 1 h at room temperature. Protein bands were visualized using the ECL system, and the optical density of the protein bands was quantified using Image J software.