Image lab software for image acquisition

A native polyacrylamide gel (15%) was used to evaluate the aggregation state of the α-synuclein. Samples (2 μg) were added with 10 μL of sample buffer (0.187 M Tris-HCl pH 6.8, 30% Glycerol, 80 μg/mL bromophenol blue). The running buffer consisted of Glycine (14.4 g/L) and Tris (3 g/L). Subsequently, the gel is stained with Coomassie Blue and decolorized in 10% acetic acid, 70% methanol, and 20% distilled H₂O. Chemidoc™ XRS detection system equipped with Image Lab Software for image acquisition (BioRad) was employed.

Cells (5 × 10⁵) were lysed in RIPA buffer (0.1% sodium dodecyl sulfate, 1% NP-40 and 0.5% sodium deoxycholate in PBS at pH 7.4) supplemented with a protease and phosphatase inhibitor cocktail. The proteins (100 μg) were loaded onto SDS-PAGE gels and electrophoretically transferred to nitrocellulose membranes (Amersham Bioscience). After blocking with 5% non-fat dry milk in TRIS buffer saline-Tween 20, the blots were incubated overnight at 4 °C with either 1:1000 anti-lamin A/C (Cell Signaling Antibodies) or 1:1000 anti-p21^Cip (Abcam) or 1:1000 anti-p53 (Abcam) primary antibodies, and then with appropriate horseradish peroxidase-conjugated secondary antibody. Proteins were detected using Enhanced Chemiluminescence reagents (ECL, Promega) and chemiluminescence was detected with a Chemidoc XRS detection system equipped with Image Lab Software for image acquisition (Bio-Rad). The quantification of protein bands was performed by determining the relative optical density using ImageJ software (National Institutes of Health, Bethesda, MD).

Five micrograms of proteins from follicular fluids were separated by 15% SDS-PAGE and electrotransfered to a nitrocellulose membrane (Amersham Bioscience, Buckinghamshire, UK). Membranes were blocked with 5% milk for 1 h at room temperature and incubated overnight at 4 °C using the following primary antibodies: 1:100 Anti-SOD2 (sc-130345) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), 1:100 Anti-Nrf2 (sc-365949) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), 1:100 Anti-NQO1 (sc-32793) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA); subsequently were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies. The signals were visualized by ECL™ Western Blotting Detection Reagents (Amersham Bioscience, Buckinghamshire, United Kingdom). All blots were imaged on Chemidoc™ XRS detection system equipped with Image Lab Software for image acquisition (BioRad, Hercules, California, USA) and processed using GelAnalizer 19.1 software (www.gelanalyzer.com by Istvan Lazar). Specific immunoreactive bands were normalized to total protein by staining membranes with Ponceau S solution (Sigma, Saint Louis, MO, USA). All experiments were replicated three times.