Fig. 3
Inhibition of JAK/STAT3, but not PI3K/Akt or MEK/ERK1/2 signaling pathways, prevents OSM-induced reduction of 3H-d-aspartate uptake in primary cortical astrocytes. a Shows the effect of 2 h pre-treatment with selective inhibitors of PI3K (LY294002, 25 μM), MEK1/2 (U0126, 5 μM), and JAK (AG490, 25 μM) on activation of Akt, ERK1/2, or STAT3 proteins, respectively, in OSM-treated (10 ng/mL for 1 h) astrocytes culture. Activation of different signaling proteins was evaluated by Western blot using antibodies that selectively detect the phosphorylation at Ser473, Thr202/Tyr204, and Tyr705 residues of Akt, Erk1/2, and STAT3, respectively. Total amounts of each protein were detected using appropriate antibodies (see the “Methods” section); β-actin served as loading control. b Shows the effect of OSM (10 ng/mL), LY294002 (25 μM), U0126 (5 μM), and AG490 (25 μM) treatments for 24 h on the viability of cultured cortical astrocytes. Cell viability was measured using a colorimetric MTT assay. OD measurements were made at 570 nm, with a blank correction made at 630 nm. Data are presented as percentage of untreated (control) astrocytes; each bar represents the average of four independent experiments done in quadruplicates. *p < 0.01. c Shows the effect of pre-treatment with LY294002 (25 μM), U0126 (5 μM), and AG490 (25 μM) on 3H-d-aspartate uptake in untreated and OSM-treated (10 ng/mL, for 24 h) astrocyte cultures. Data are normalized to untreated controls and presented as mean ± SEM; **p < 0.001 (compared to untreated control); ## p < 0.001 (compared to OSM); n = 8; one-way ANOVA