Te77xp semidry blotter

Chondrocyte pellets were dissolved in citrate-EDTA buffer (55 mM/5 mM; pH 6.8) for 10 min at 37 °C and centrifuged at 10 K rpm for 15 s. Then, the cell pellet was washed five times with PBS and resuspended in 200 μL of SDS-NuPAGE buffer for NuPAGE gel (Invitrogen, Grand Island, NY, USA) running. Immunoblotting was performed using monoclonal antibodies against Type Ⅰ collagen, Type Ⅱ collagen (1:1000 dilution; EMD Millipore, Billerica, MA, USA), MMP-13 (Abcam, Cambridge, MA, USA) TGF-β (1:1000 mg/mL; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and PPAR-γ (Abcam, Cambridge, MA, USA). The loading control, β-actin, will be detected using anti-β-actin antibody (1:10,000 dilution; Abcam, Cambridge, MA, USA). Secondary antibodies tagged horseradish peroxidase (HRP) (rabbit for TGF-β 1:20,000 dilution and mouse for type Ⅱ collagen and β-actin, 1:5000 dilution) was used. Proteins separated in the gel and the gel were transferred to nitrocellulose membrane (GE Healthcare Life Sciences, Pittsburgh, PA, USA) using TE77XP semidry blotter with 10 V for 3 h (Hoefer, Inc., Holliston, MA, USA). Protein band signals on blots were detected on Amersham Hyperfilm which was enhanced chemiluminescence (GE Healthcare Life Sciences, Pittsburgh, PA, USA) using SuperSignal West Pico Chemiluminescent Substrate kit (Thermo Fisher Scientific, Rockford, IL, USA).

Chondrocyte pellets were stored at −80 °C until assayed. Frozen samples were pulverized under liquid nitrogen and placed in a homogenization buffer (10 mM phosphate buffer, 250 mM sucrose, 1 mM EDTA, 0.1 mM phenylmethanesulfonyl fluoride or phenylmethylsulfonyl fluoride, and 0.1% [v/v] Tergitol, pH 7.5). Homogenates were centrifuged at 27,000g for 10 min at 4 °C to harvest supernatant for immunoblotting with antibodies. Immunoblotting was performed using primary monoclonal antibodies against PPAR-δ (1:2,000 dilution; Abcam, Cambridge, MA, USA), type II collagen (1:1,000 dilution; EMD Millipore, Billerica, MA, USA), TGF-β (1:1,000 μg/mL; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and β-actin (1:10,000 dilution; Abcam) and secondary antibodies tagged horseradish peroxidase (HRP) (rabbit for PPAR-δ and TGF-β, 1:20,000 dilution, mouse for type II collagen and β-actin, 1:5,000 dilution). Cell pellets were processed for NuPAGE gel (Invitrogen). Proteins separated in the gel were transferred to nitrocellulose membrane (GE Healthcare Life Sciences, Pittsburgh, PA, USA) using TE77XP semidry blotter with 10 V for 3 h (Hoefer, Inc., Holliston, MA, USA). Protein band signals on blots were detected on Amersham Hyperfilm enhanced chemiluminescence (GE Healthcare Life Sciences) using SuperSignal West Pico Chemiluminescent Substrate kit (Thermo Fisher Scientific, Rockford, IL, USA).