Western blot for Nrf2 and HO-1

Cell lysates were prepared and quantified as mentioned above and then electrophoresed and transferred onto nitrocellulose membrane. In case of Nrf2, 5% bovine serum albumin (BSA) was used for blocking the membrane followed by probing with 1:1000 diluted monoclonal Nrf2 antibody and 1:2000 diluted goat anti-mouse HRP-conjugated secondary antibody. Detection was done by ECL and chemiluminescence was recorded on X-ray film. Membrane was stripped using stripping buffer (10% SDS, 0.5M Tris-Cl, β-mercaptoethanol) at 55°C for 30 min and washed 5 times using phosphate-buffered saline. For HO-1, 5% BSA was used for blocking the membrane followed by probing with 1:500 diluted monoclonal HO-1 antibody and 1:2000 diluted goat anti-mouse HRP-conjugated secondary antibody followed by detection. Membrane was restripped and probed for β-actin using 5% nonfat dry milk for blocking, 1:1000 diluted monoclonal β-actin antibody followed by the above given procedure.