Nadph quantification kit

Embryos were dechorinated with pronase at 24 hpf and treated with 100 μg mL⁻¹ of chloroquine and/or AG1 (1 μM) by directly adding the compounds to the well. For ROS measurements, the embryos were incubated with compounds for 5 h and then the ROS-detecting reagent (CM-H₂DCFDA) was added at a final concentration of 500 ng mL⁻¹ to the well and incubated for 3 h. One embryo was placed to each well of a black, opaque 96-well plate. The florescence was analyzed with excitation/emission at 485/525 nm. After ROS assays, embryos at about 32 hpf were pooled and lysed in buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.1% NP-40 and protease inhibitor cocktail, which was followed by three cycles of freeze and thawing in liquid nitrogen. The lysate was centrifuged at 18,800 × g at 4 °C for 15 min. Total protein concentration in the supernatants (total lysate) was determined by the Bradford method. 10 μg of total lysate was used for enzymatic assay. 50 μg of total lysate was used to measure NADPH levels using a NADPH quantification kit (Biovision), according to the manufacturer’s instructions. 50 μg of total lysate was loaded onto 10% SDS–PAGE gels, and the protein levels were examined by Western blot using anti-G6PD antibody (Abcam (G6PD: AB87230)).