Pv3227

Doses of 500 mg/kg 2DG (D8375, Sigma-Aldrich), 4 mg/kg CCCP (C2759, Sigma-Aldrich), 100 mg/kg meclizine (B1786, Apexbio), and 50 mg/kg or 5 µmol/kg ATP (PV3227, Thermo Fisher) were injected intraperitoneally.
We injected 500 mg/kg 2DG or 4 mg/kg CCCP immediately after surgery to investigate the numbers of surviving RGCs after inhibition of glycolysis and oxidative phosphorylation. For the analysis of glucose metabolism (Fig. 5), mice were injected with the same dose of 2DG (500 mg/kg)/iodoacetic acid (60 mg/kg)/oligomycin (0.5 mg/kg)/CCCP (4 mg/kg) 1 h prior to euthanasia.

Radioactive in vitro kinase assays for ERK3 proteins were done as previously described [18 (link)]. Briefly, each in vitro kinase assay reaction contained 40 nM ERK3 protein, 1 µg substrate, 5 μCi (γ-³²P)-ATP (Perkin Elmer, Waltham, MA, USA, catalog # NEG002Z) and 25 μM cold ATP (ThermoFisher, catalog # PV3227). The reactions were carried out at 30 °C for 30 min and then stopped by SDS sample buffer and boiling. Proteins were resolved by SDS-PAGE, stained with Coomassie blue solution and visualized by autoradiography. The quantification of substrate phosphorylation was determined by calculating the ratio of the band intensity of phosphorylated substrate in the autoradiograph over that of the corresponding total substrate protein in the Coomassie-stained gel.