Nu page 4 12 sds page gels

Cell lysates (10 µg protein) were separated on Nu-Page 4–12% SDS-PAGE gels (Life Technologies, Paisley, UK), transferred to a nitrocellulose membrane, andthen blocked using PBS/Tween bufferand nonfat dried milk (w/v 1%) solution for 1 h. Following this membranes were incubated overnight (15 h, 4 °C) with primary antibodies versus superoxide dismutase 1 (SOD1, 1:1000) (Cat. No.ADI-SOD-100, Enzo Life Sciences, Devon, UK), superoxide dismutase 2 (SOD2, 1:1000) (Cat. No. ADI-SOD-111, Enzo Life Sciences, Devon, UK), COL2A1 (1:1000) (Cat. No. sc-7764, Santa Cruz Biotechnology, Hiedelberg, Germany) or SOX9 (1:1000) (Cat. No.AB5535 Millipore, Herts, UK) with α-tubulin (1:1000) (Cat. No.ab4074, Abcam, Cambs, UK) used as loading control. Membranes were washed thrice using PBS/Tween and incubated with horseradish peroxidise (HRP)-conjugated secondary antibody (1:2000, SourceBioscience, Notts, UK) (1 h, 20 °C) with bands detected using a chemiluminescence detection kit (Western Lightning Plus, Bucks, UK). Bands were imaged using the VisionWorksLS image acquisition software package and band densities were analyzed using ImageJ 1.42 software and normalized to loading control.

Cells were rinsed with PBS and lysed using the Laemmli sample buffer (50 mM Tris pH 6.8, 2% SDS, 0.025% Bromophenol Blue, 10% glycerol, 5% BME). Lysates were boiled for 20 min, resolved using NuPAGE 4–12% SDS–PAGE gels (Life Technologies) and transferred to PVDF membranes (Millipore). Membranes were blocked using the Superblock T20 (TBS) Blocking Buffer (#37536 from Thermo Fisher Scientific), probed with primary antibodies overnight at 4 °C, and horseradish peroxidase (HRP) conjugated secondary antibodies at room temperature for 1 h. The immune complexes were detected by SuperSignal^TM West Dura Substrate (#34075 from Thermo Fisher Scientific).