Fig. S3.

Xanthine oxidase, nitric oxide synthase, or NADPH oxidases do not account for the high rate of ROS production by astrocytes. (A) Inhibition of xanthine oxidase, a well-known nonmitochondrial O₂^•− source, using allopurinol at a wide range of effective concentrations, did not alter the rate of H₂O₂ production in mouse primary astrocytes. (B) Nitric oxide synthase (NOS) inhibition with nitro-l-arginine methyl ester (NAME) did not alter H₂O₂ production in astrocytes. (C) mRNA relative abundances of the nonmitochondrial NADPH oxidase (NOX)-1 (NOX1) and NOX2, and mitochondrial NOX4, well-known sources of superoxide, in neurons and astrocytes. (D) Inhibition of NOXs using VAS2870 at a wide range of effective concentrations did not alter the rate of H₂O₂ production in astrocytes. (E) siRNA-mediated knockdown of the NOXs assembly protein, p22^phox, was confirmed by RT-qPCR and Western blotting in astrocytes. The rate of H₂O₂ production was unaltered in p22^phox-knockdown astrocytes. Data are the mean values ± SEM from n = 3–4 independent culture preparations (Student’s t test; ANOVA post hoc Bonferroni). *P < 0.05; n.s., not significant.