Anti nrf2 16396 1 ap

The ipsilateral striatum of the mouse brain and cultured cells were homogenized in enhanced RIPA lysis buffer (AR0101, Boster, China) and 10% protease inhibitor cocktail (04693132001, Roche, USA) by sonication. The protein concentration was determined with the BCA protein assay kit (23221, Thermo). The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (1620177, Bio-Rad, USA), which were blocked in 5% non-fat milk before being incubated with primary antibodies overnight at 4°C. The primary antibodies were anti-LRRC8A (sc-517113, Santa Cruz, USA), anti-AMPK (5832S, Cell Signaling Technology, USA), anti-phospho-AMPK (Thr172; 50081S, Cell Signaling Technology, USA), anti-Nrf2 (16396-1-AP, Proteintech, USA), anti-lamin B1 (MA1-06103, Invitrogen), anti-CD36 (sc-7309, Santa Cruz), anti-α-tubulin (ab52866, Abcam), and anti-β-actin (sc-47778, Santa Cruz). The membranes were washed with TBST buffer and then incubated with IgG HRP-conjugated secondary antibodies (anti-mouse, 7076S, Cell Signaling Technology, or anti-rabbit, 7074S, Cell Signaling Technology) for 1h at room temperature. Immunoreactive bands were visualized by using chemiluminescent HRP substrate (90719, Millipore, USA). The bands were scanned and analyzed with ImageJ software.