4.5. Western Blot Analysis for Nrf2 and PPARγ

Protein from nuclear fractions was used for expression characteristics of NRF2, and cytosolic fractions were used for PPARγ by western blot (WB). Nuclear and cytosolic extractions from explants were performed using the kit instructions (NE-PER cat #78833 from ThermoFisher Scientific, Waltham, MA, USA). Briefly, explants were homogenized using a Dounce homogenizer (Fisher Scientific, Atlanta, GA, USA) in the provided CER I buffer containing freshly added protease inhibitors. The homogenates were then vortexed and incubated on ice for 10 min. Cold CER II buffer was then added to the tube, and the homogenates were vortexed for 5 s and incubated on ice for 1 min followed by centrifugation at 16,000× g for 5 min. The supernatant containing the cytoplasmic extracts were removed and stored on ice. The pellets containing the nuclei were resuspended in cold NER buffer containing freshly added protease inhibitors, and vortex vigorously on and off for 40 min. The tubes were then centrifuged at 16,000× g for 10 min, and the supernatants containing the nuclear fraction were transferred into new tubes and kept on ice. Protein concentrations were estimated in both cytosolic and nuclear fractions (BCA Protein Assay Kit, Thermo Scientific, Waltham, MA, USA) and 20ug of protein was used for each sample to run the western gels as described previously [32]. Immunoreactive proteins were visualized using chemiluminescence reagents ECL WB detection system (Amersham Piscataway, NJ, USA). The anti-human and anti-mouse antibodies used for WBs were as follows: Nrf2 (1:1000, ab62352, Abcam, Cambridge, UK), PPARγ (1:1000,ab19481, Abcam, Cambridge, UK), nuclear protein Histone (D1H2 1:1000, Rabbit mAb, Cell Signaling Technology Danvers, MA, USA) and β-actin (1:15,000, Sigma- Aldrich, St. Louis, MO, USA). The relative levels of the proteins in the specific bands were normalized with either Histone (for Nrf2) or β-actin (for PPARγ) expressions in the same samples, and expressions were densitometrically determined using the Bio-Rad-Image Lab software (version 6.0, Bio-Rad, Hercules, CA, USA).