Anti gclc

Primary antibodies used for Western blotting are listed as follows, the dilutions are indicated in the list. Anti-PGC1 alpha (ab191838, dilution 1:1000), Anti-NOX2/gp91phox (ab129068, 1:500), Anti-GCLC (ab190685, 1/500), Anti-PRDX3 (ab73349, 1:1000), Anti-p62 (ab91526, 1:1000), Anti-Citrate synthase (ab129095, 1:1000), Anti-GAPDH (ab8245, 1:2000) from Abcam (Cambridge, UK); Anti-AMPKα (#2532, 1:400), Anti- Phospho-AMPKα^Thr172 (#2535, 1:400), Anti-MFN2 (#9482, 1:500), Anti-ULK1 (#8054, 1:200), Anti-Phospho-ULK1^Ser555 (#5869, 1:250), Anti-LC3B (#43566, 1:250), from Cell Signalling (Cell Signalling Technology, Danvers, MA, USA); Anti-SOD2 (ADI-SOD-111, 1:2000) from Enzo Life Sciences (Farmingdale, New York, USA). Primary antibodies Anti-MYH I (BA-D5, 1:200), Anti-MYH IIb (BF–F3, 1:250), and Anti-MYH IIa (SC-71, 1:250) from Developmental Studies Hybridoma Bank (Iowa, USA) and Wheat Germ Agglutinin (WGA), CF®405S Conjugate (29,027–1, 1:200, Generon, Slough, UK) for membrane staining were used for immunohistochemistry.

Total proteins were obtained from the treated BEND cells with a protein extraction kit (Solarbio, Beijing, China). The protein concentration was detected by the BCA method (Solarbio, Beijing, China). The proteins (50 μg) were heated in loading buffer at 95 °C for 5 min. Each protein sample was subjected to SDS-PAGE using a gel preparation kit (Solarbio, Beijing, China) to separate proteins, then proteins were transferred to the PAGE membrane and blocked with 5% skim milk at 4 °C overnight. After rinsing with TBST on a shaker, membranes were hybridized for 90 min at room temperature with anti-Nrf2 (1:2000, Abcam, Cambridge, UK), anti-GCLC (3:1000, Abcam), anti-HO-1 (3:1000, Abcam), anti-NQO-1 (1:2000, Novus, Centennial, CO, USA), anti-Mn-SOD (1:5000, Abcam), and anti-β-actin (1:1000, Cell Signaling) antibodies, respectively. After rinsing for 1 h, membranes were incubated with corresponding horseradish peroxidase-conjugated secondary antibodies for 90 min at room temperature. After rinsing again for 1 h, proteins were visualized using Enhanced Chemiluminescence (ECL) system. Finally, an image-analysis system was used to analyze the density of these target proteins.

ACR was purchased from Sigma-Aldrich (St. Louis, MO, USA). Tris-HCl, EDTA-Na2, and sucrose were purchased from Biotopped (Beijing, China). GSH, AST, ALT, MDA, SOD, Bradford protein assay kit, and Mitochondrial ATPase were bought from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). PVDF membranes were purchased from Merck Millipore (Burlington, MA, USA). Anti-Nrf2 antibodies were purchased from Proteintech (Wuhan, China). Anti-HO-1, anti-NQO1, anti-GCLC, anti-GCLM, and anti-β-actin were obtained from Abcam (Cambridge, UK). Trizol, PrimeScript™ RT Master Mix wi, and TB Green Premix Ex Taq™ II were purchased from Takara (Kusatsu, Japan). All chemicals employed in this work were analytical grade. The LRP sample preparation was according to the method of Gao et al. [25 (link)]. LRP was composed of nine phenolic compounds, among which rutin was the most abundant at 1013.05 ± 33.70 mg/100 g, followed by p-coumaric acid, catechin, caffeic acid [25 (link)].

According to the manufacturer’s instructions, the protein extracts of nucleus and cytoplasm (three mice in each group) were prepared using nuclear and cytoplasmic protein extraction kit (Beyotime Institute of Biotechnology, Shanghai, China). The total protein was assayed using BCA protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China). Proteins (50 μg) were subjected to 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After blocked with 5% nonfat milk for 1 h, the membranes were incubated overnight at 4 °C with the primary antibodies for cytosolic fractions: rabbit anti-TH antibody (1:1000, Millipore Corporation), rabbit anti-phospho-PI3K (1:1000, Cell Signaling Technology), rabbit anti-PI3K (1:1000, Cell Signaling), rabbit anti-phospho-AKT (1:1000, Cell Signaling), rabbit anti-AKT (1:1000; Cell Signaling), anti-GCLC (1:1000, Abcam), anti-GCLM (1:2000, Abcam), anti-Heme Oxygenase 1 (1:2000, Abcam), anti-Keap1 (1 μg/ml, Abcam), rabbit anti-β-Actin (1:1000; Cell Signaling), and the primary antibodies for nuclear fractions: anti-Lamin B1 (0.1 μg/ml, Abcam), anti-Nrf2 (1:1000, Abcam). The membranes were incubated with the horseradish peroxidase-labeled secondary antibody (1:2000). Bands were investigated using the ECL detection reagents (Beyotime Institute of Biotechnology, Shanghai, China).