Fig. 8

ARNT2 protein is localized to both neurons and astrocytes in neuronal-enriched cortical cultures. a Immunocytochemistry for ARNT2 reveals negligible ARNT2 staining in the Bend3 mouse immortalized brain microvascular endothelial cell line. Using threshold sets by the staining intensity in negative cells as described in the “Methods” section and Additional file 3, cells were divided according to staining intensity: negligible to ++++. The majority of MAP2⁺ neurons in the culture showed low to moderate staining for ARNT2; staining was also detected in the few contaminating GFAP⁺ astrocytes which demonstrated a reactive phenotype. One of three representative experiments is shown where eight regions from each of two duplicate wells were analyzed. b Following exposure to 100 μM H₂O₂, the entire enriched population as well as MAP2⁺ neurons and GFAP⁺ astrocytes examined individually demonstrated increases in ARNT2 staining intensity at 1 h; the same decreases after longer exposure observed with western blotting to baseline levels and below were replicated by immunocytochemistry. Points represent eight biological replicates, with two technical replicates each, where lines and whiskers indicate mean and standard deviation. p ≤ 0.05*, p ≤ 0.01**, p ≤ 0.001***, repeated measures ANOVA on ranks with Tukey’s multiple comparison test to unstimulated