Protease inhibitor mixture 1x

Drug-treated SK-HEP1 cells were lysed with radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor mixture (1X) (Roche, IN, USA) and 2mM Phenyl methyl sulfonyl fluoride (Sigma Aldrich). Immunoblotting with equal amounts of proteins (30ug) was carried out as reported previously [70 (link)] (antibodies listed in S1 Table).

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 (link)].