IFN-β mRNA expression and effect of IFN-β neutralization on NO production and STAT-1 activation in BALB/c and C57BL/6 macrophages.

<p>Peritoneal macrophages (5×10⁶) were stimulated with 1 µg/mL LPS for the indicated periods of time. Total RNA was extracted and IFN-β mRNA levels were determined by Real Time RT-PCR. The relative levels of mRNA expression were calculated by reference to the β-actin expression in each sample, using the 2^−ΔΔCt method (A). Alternatively, cells (1×10⁵) were stimulated for 24 h (B) or for the indicated periods of time (C–E) with 1 µg/mL LPS in the presence or absence of the indicated concentrations (B) or with 200 U/mL of polyclonal anti-IFN-β (C–E). Culture supernatants were analyzed for NO⁻₂ levels using the Griess reaction, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098913#s2" target="_blank">Materials and Methods</a>. Purified normal rabbit IgG was used as an isotype control. (B) represents the dose-response of one experiment and (C) represents the time course of a second one, out of three independent experiments. Values represent the mean of samples assayed in triplicate. (D and E) represents STAT-1 activation as analyzed by Western blot of total protein extracts of C57BL/6 peritoneal macrophages (3×10⁵) using antibodies specific for pSTAT-1 (Y701) and β-actin. The levels of pSTAT-1 (E) were expressed as ratios of the signal intensity of the bands normalized to that of β-actin. Data are representative of three independent and reproducible experiments. Additional experiments to illustrate the variability in the results are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098913#pone.0098913.s004" target="_blank">Fig. S4</a>.