Anti ampk 5832s

Total proteins were extracted by RIPA. Proteins were separated by SDS-PAGE and electro-transferred onto PVDF membranes. The membranes were soaked in buffer containing 5% BSA for 1 h and then incubated with primary antibodies including anti‐p-mTOR (5536s, 1:1000, Cell Signaling Technology), anti‐mTOR (2983s, 1:1000, Cell Signaling Technology), anti‐p-PI3K (bs-6417R, 1:1000, Bioss), anti‐PI3K (bs-10657R, 1:1000, Bioss), anti‐p-AMPK (2531S, 1:1000, Cell Signaling Technology), anti‐AMPK (5832S, 1:1000, Cell Signaling Technology), anti‐p-AKT (Sc-293125, 1:1000, Santa), anti‐AKT (Sc-5298, 1:1000, Santa), anti‐PGC1α (bs-7535R, 1:1000, Bioss), anti‐LC3B (ab51520, 1:5000, Abcam), anti‐ZO-1 (61–7300, 1:1000, Invitrogen), anti‐Occludin (71–1500, 1:1000, Invitrogen), or anti‐Claudin-5 (34–1600, 1:1000, Invitrogen), at 4 °C overnight. Thereafter, the membranes were washed with Tris‐buffered saline Tween buffer and incubated with HRP‐rabbit (ab191866, 1:5000, Abcam) or HRP‐mouse (bs-0296G-HRP, 1:1000, Bioss) conjugated secondary antibody for 1 h at room temperature (RT). An Odyssey Infrared Imaging System was used to scan the membranes for further analysis. β-actin (CL594-66009, 1:1000, Proteintech) was used as loading control. Band intensities were quantified using Image J software.

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.