Figure 3
NECA-induced leukemia inhibitory factor (LIF) expression and secretion levels in primary astrocytes are dependent on adenosine A 2B receptor activation. Primary cortical astrocytes were treated with the adenosine analog, NECA (1 μM) or a selective adenosine A2A 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 A2A receptor antagonist (ZM 241385, 250 nM) or adenosine A2B 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 A2B 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.