Basal and NECA-induced leukemia inhibitory factor (LIF) expression and secretion levels in primary astrocytes are dependent on NF-κB activation. (A) shows Western blot analysis of primary cortical astrocytes treated without or with NECA (1 μM; for 0.5, 1, 2 and 4 hours) to detect phosphorylation at Ser536 of NF-κB p65 (RelA) proteins. Where indicated, cells were pre-treated with selective inhibitor of NF-κB (BAY 11–7082, 10 μM) for 2 hours prior to NECA stimulation. β-actin served as a loading control. Subsequently, astrocytes were treated without or with NECA (1 μM) for 2 hours (for real-time PCR) and 4 hours (for Western blot and ELISA), in presence or absence of BAY 11–7082 (10 μM, added 2 hours prior to NECA stimulation). (B) shows real-time PCR analysis of LIF gene expression (relative to GAPDH). Data are normalized to the control and presented as Mean ± SEM of three independent experiments. P < 0.05. (C) shows Western blot analysis to detect LIF protein levels. β-actin served as a loading control. (D) shows a representation of sandwich ELISA experiment performed to detect LIF content in supernatants of cultured astrocytes. Each bar corresponds to the mean concentration of LIF in triplicate samples; error bars indicate SEM. Observations were confirmed by repeating the experiments two additional times. P < 0.05. NECA, 5′-N-ethylcarboxamide.