Western blotting and co-immunoprecipitation (co-IP)

The detailed protocols of Western blotting were previously reported [30, 31]. In brief, lysate proteins were separated by SDS-PAGE gels [32], transferred to PVDF blots(Millipore, Shanghai, China). The blots were blocked and incubated with the designated primary and secondary antibodies. An ECL reagent kit (Pierce, Shanghai, China) was applied to detect the protein band under X-ray films. Data quantification was carried out by an ImageJ software (NIH). For the co-IP studies, the quantified protein lysates (1, 000 μg for each treatment) were pre-cleared and incubated with anti-Keap1 antibody [33]. Keap1-Nrf2 complex was captured by the G-Sepharose (“Beads”, Sigma), tested by Western blotting. Testing nuclear fraction lysates was described in our previous studies [30, 31]