Figure 5

NECA-induced leukemia inhibitory factor (LIF) expression and secretion levels in primary astrocytes are blocked by protein kinase C (PKC) inhibitor, but not by protein kinase A (PKA) inhibitor. Primary cortical astrocytes were treated without or with NECA (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 specific inhibitors of PKA (KT 5720, 250 nM) or PKC (Ro 31–8220, 250 nM), prior to NECA stimulation. (A) 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. (B) shows Western blot analysis to detect LIF protein levels. β-actin served as a loading control. (C) 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.