Anti ampk

The reagents used included 3MA (Sigma-Aldrich, S2767), rapamycin (Sigma-Aldrich, S1039), bafilomycin A1 (Sigma-Aldrich, SML1661), VEGF (PeproTech, 100-20A), N-Nitro-L-Arginine Methyl Ester (L-NAME, Sigma-Aldrich, N5751), Diethylenetriamine/nitric oxide adduct (2,2′-(Hydroxynitrosohydrazono)bis-ethanimine DETA/NO, NO donor, Sigma-Aldrich, D185).
The antibodies used included anti-Col I (Proteintech, 14695-1-AP), anti-α-SMA (Boster, BM0002), anti-vWF (Santa Cruz, SC-365712), anti-CD31 (Santa Cruz, SC-46694), anti-Cav-1 (Abcam, ab17052), anti-Cav-1 (Abcam, ab2910), anti-LC3 (Abcam, ab48394), anti-p-VASP (Ser157) (CST, 84519), anti-VASP (CST, 3132S), anti-p-eNOS (Ser1177) (CST, 9570), anti-eNOS (Abclonal, A1548), anti-p-PI3K (Tyr458) (CST, 4228), anti-PI3K (CST, 4249), anti-p-AKT (Ser473) (CST, 4060), anti-AKT (CST, 2938), anti-p-MTOR (Ser2448) (CST, 5536), anti-MTOR (CST, 2983), anti-GLUT3 (Abcam, ab41525), anti-AMPK (Proteintech, 10929-2-AP), anti-ULK1 (Proteintech, 20986-1-AP), anti-GAPDH (Proteintech, 60004-1), and anti-β-actin (Proteintech, 60008-1). DAPI (Sigma-Aldrich, D9542), FITC-labeled goat anti-rabbit IgG (H+L) (Beyotime, a0562), Cy3-labeled goat anti-mouse IgG (H+L) (Beyotime, a0521), and phallotoxins (Thermo, F432) were also used.

Fouty-eight rats were used for the immunoblot analysis. Optic nerves were isolated 1 week after intravitreal administration and homogenized in protein extraction buffer. After being centrifuged, the supernatant was used for protein concentration measurement and processed in sample buffer (Bio-Rad Laboratories). Equal amounts of each sample were applied to SDS-PAGE gels (Bio-Rad Laboratories), ran, and transferred to an enhanced chemiluminescent membrane. Primary antibodies used were anti-p-AMPK (1:200, Thr172; Sigma-Aldrich), anti-AMPK (1:200; Proteintech, Rosemont, IL, USA), and anti-β-actin (1:5000; Sigma-Aldrich). After washing, the membranes were reacted with peroxidase-labeled secondary antibodies (1:5000). An electrochemiluminescence (ECL) system was used for visualization of immunoblotting.

The chemical reagents A769662, Rapamycin and Z-VAD(OMe)-FMK were purchased from Cell Signaling Technology (USA).
Bafilomycin A1 (Baf A1), chloroquine (CQ) and deferoxamine mesylate salt (DFO) were purchased from Sigma-Aldrich (USA).
Anti-ARA70 and anti-ACSL4 antibodies were purchased from Santa Cruz Biotechnology (USA).
Anti-p62/SQSTM1, anti-AMPK and anti-pAMPK antibodies and horseradish peroxidase (HRP)-conjugated secondary antibody were purchased from Proteintech (USA).
4′,6-diamidino-2-phenylindole(DAPI), RIPA lysate buffer, SA-βgal staining kit,
BCA assay kit, ROS assay kit, JC-1 assay kit and GSH assay kit were purchased from Beyotime (Shanghai, China).
Anti-LC3B, anti-Ferritin Heavy Chain, Anti-Glutathione Peroxidase 4, anti-beta Actin and anti-GAPDH antibodies were purchased from Abcam (USA).
Real-time quantitative PCR (RT-qPCR) amplification primers for β-actin, AMPK, p-AMPK, GPX4, ACSL4 and SLC7A11 were synthesized by TsingkeBiotechnology (Beijing).
Cell Counting Kit-8 (CCK-8), malondialdehyde (MDA) assay kit and FerroOrange assay kit were purchased from Dojindo (Japan).
Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Gibco (USA). Fetal bovine serum was purchased from Biological Industries (ISL).