To obtain whole cell lysates for immunoblotting analysis, cells were washed twice in 1 ml PBS and lysed with buffer containing 10 mM Tris HCl (pH 7.5), 0,9% NP-40, 0,1% SDS, 1 mM Pefabloc, and homogenized by passing through a 25-gauge needle ten times. Insoluble material was removed by centrifugation at 14,000 rpm for 20 min at 4 °C. The cytoplasmic and nuclear extracts were performed through the instructions of Nuclear Extract Kit (Active Motif, 40010). Protein concentrations were determined by BCA method.
Following the heat treatment and lysis of HT22 cells, 30 μg of whole cell lysates and nuclear lysates in reducing Laemmli-buffer (0.25 M Tris pH 6.8, 8% SDS, 40% glycerol, 0.03% bromophenol blue) were denatured at 95 °C for 5 min and applied to SDS-PAGE of 12% (w/v) acrylamide, followed by electrophoresis and blotted onto nitrocellulose membrane according to standard procedures. Immunodetections were performed with the following antibodies: anti-Nrf2 (D1Z9 C), anti-HSP90 (C45G5), anti-HSP70 (D69), anti-HSP40, anti-HO-1 (P249), anti-proteasome β5, anti-GSTα and anti-β-actin at 1:1000 dilutions. After exposure to peroxidase-coupled secondary antibodies (Bio-Rad) at 1:5000 dilution, membranes were developed using Lumi-Light western blotting substrate (Cell Signaling). Blots were visualized using X-ray film and quantified by densitometry using Image J software.
Following the heat treatment and lysis of HT22 cells, 30 μg of whole cell lysates and nuclear lysates in reducing Laemmli-buffer (0.25 M Tris pH 6.8, 8% SDS, 40% glycerol, 0.03% bromophenol blue) were denatured at 95 °C for 5 min and applied to SDS-PAGE of 12% (w/v) acrylamide, followed by electrophoresis and blotted onto nitrocellulose membrane according to standard procedures. Immunodetections were performed with the following antibodies: anti-Nrf2 (D1Z9 C), anti-HSP90 (C45G5), anti-HSP70 (D69), anti-HSP40, anti-HO-1 (P249), anti-proteasome β5, anti-GSTα and anti-β-actin at 1:1000 dilutions. After exposure to peroxidase-coupled secondary antibodies (Bio-Rad) at 1:5000 dilution, membranes were developed using Lumi-Light western blotting substrate (Cell Signaling). Blots were visualized using X-ray film and quantified by densitometry using Image J software.