Nrf2 protein expression by western blot and subcellular localization by Immunofluorescence

Cells were washed with ice-cold PBS, and the whole cell lysates were prepared in Radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitor mixture (1X) (Roche Applied Science, IN, USA) and 2mM Phenyl methyl sulfonyl fluoride (Sigma-Aldrich) (Sigma–Aldrich, Bangalore, India). Protein concentration was estimated using Bradford method. Total proteins (50μg/lane) were resolved in 10% SDS-Polyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 10% non-fat dry milk powder in TBS containing 0.1% Tween20 and then incubated with Anti-Nrf2 antibody. β –Actin was used as loading control. The bands were visualized using a chemiluminescence ECL system (Super Signal West Femto, Thermo Scientific). The intensity of protein bands was quantified by FluorChem E system using Digital Darkroom software. Subcellular localization of Nrf2 was determined by immunofluorescence as previously described [27].