Figure 3.

NECA-induced leukemia inhibitory factor (LIF) expression and secretion levels in primary astrocytes are dependent on adenosine A_2Breceptor activation. Primary cortical astrocytes were treated with the adenosine analog, NECA (1 μM) or a selective adenosine A_2A receptor agonist (CGS 21680, 1 μM) for 2 hours (for real-time PCR) or 4 hours (for Western blot and ELISA). Where indicated, astrocytes were pre-treated for 30 minutes with selective adenosine A_2A receptor antagonist (ZM 241385, 250 nM) or adenosine A_2B receptor antagonist (MRS 1754, 250 nM), prior to NECA stimulation. (A and B) show real-time PCR analyses of LIF gene expression (relative to GAPDH) in wild-type and A_2B knock-out astrocytes, respectively. Data are normalized to the control and presented as Mean ± SEM of three independent experiments. P < 0.05. (C) shows Western blot analyses to detect LIF protein levels in wild-type astrocytes. β-actin served as a loading control. (D) shows a representation of sandwich ELISA experiment performed to detect LIF content in supernatants of cultured wild-type 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.