Aggrecan antibody

Cells from different experimental conditions were lysed with ice‐cold RIPA lysis buffer and protease inhibitors. A BCA protein assay kit was used to measure the protein concentration according to the manufacturer's instructions. SDS‐PAGE electrophoresis was carried out with 20 μg per lane of lysate protein and 10% or 6% polyacrylamide gels, followed by electrophoresis onto polyvinylidene difluoride (PVDF) membranes. After the transfer, the membranes were blocked with 5% dried skimmed milk in TBST buffer (Solarbio, PH 7.6). The PVDF membranes incubated with primary antibodies in TBST, which contained 5% dried skimmed milk overnight at 4°C. The membranes were washed in TBST buffer, and then incubated with the secondary antibody for 1h at RT. Immunoreactivity was detected with the ECL substrate (Thermo Fisher Scientific). Anti-FGF9 antibody was purchased from Abcam. Collagen II polyclonal antibody was from Invitrogen, Collagen X antibody was from Abcam Technology, MMP13 polyclonal antibody was from Bioworld Technology, and Aggrecan antibody was from Affinity Biosciences. Primary antibodies (total or phospho-) specific for AKT (or Protein Kinase B), Glycogen synthase kinase 3 (GSK3), mammalian Target of Rapamycin (mTOR) were from Cell Signaling Technology. GAPDH expression was analyzed as an internal control during the Western blot analyses.