Restore tm plus stripping buffer

Nuclear and cytoplasmic proteins from the treated cells were isolated using a nuclear extraction kit (Cayman Chemicals). Protein concentration in the extracts was measured with Bradford reagent (Bio-Rad). Equal amount of proteins were subjected to 12% SDS-PAGE electrophoresis followed by Western blot analysis using specific antibodies against Nrf2, Keap1, HO1, NQO1, AMPK, histone H3, and GAPDH. The antigen-antibody complexes were detected by enhanced Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific). Membranes were stripped with Restore TM PLUS stripping buffer (Thermo Scientific) and used for reprobing with other antibodies or loading controls.

Western blot lysates were produced in RIPA buffer (Pierce #89900) supplemented with cOmplete Mini EDTA-free protease inhibitor cocktail and PhoshoStop phosphatase inhibitor tabs (Roche) and 1 mM DTT at 4 °C. Lysates were cleared by centrifugation at 16,000 x g for 25 min at 4 °C. Proteins were separated using 3–8% or 7% Tris-Acetate, or 4–12% Bis-Tris precast mini gels (Life Technologies) and transferred to PVDF membranes at 70 V for 2 h or overnight (Immobilon P, Millipore). Ponceau S staining was used to verify even transfer (Sigma Aldrich). Membranes were optionally cut to detect multiple proteins. Blocking and antibody incubations were then performed with either 5% non-fat milk or bovine serum albumin (fraction V) in PBST or TBST. Bands were visualized with HRP-conjugated secondary antibodies (DAKO) followed by the application of a chemiluminescent reagent (Thermo Scientific). Stripping with Restore^TM PLUS stripping buffer (Thermo Scientific) and reprobing was performed in some cases when the subsequent primary was of a different species; however, blots were never stripped more than once. Densitometry was performed in ImageJ on unaltered images.