Figure 2. Attenuated inflammatory response of ARF deficient macrophages to classical activation. (A) Peritoneal WT and ARF-deficient macrophages were stimulated with 200 ng/mL lipopolysaccharide (LPS) for 24 h and NO release was determined by the Griess reaction. (B) PGE₂ levels were assessed by ELISA in cells treated as in A. (C) Peritoneal macrophages from WT and ARF-deficient mice were activated for 6 h with 200 ng/mL LPS and expression of iNOS and COX-2 was evaluated by quantitative PCR. (D) Protein levels of iNOS and COX-2 were evaluated by immunoblotting after stimulation of peritoneal macrophages with 200 ng/mL LPS for the indicated time. Band intensities were quantified by densitometry, normalized to β-actin levels and represented as means ± SD of the fold change from control condition (n = 3). (E) NO release of peritoneal macrophages from WT and Arf^−/− mice after stimulation for 24 h with 50 ng/mL γinterferon γ (IFNγ). (F) iNOS induction was examined by quantitative PCR in WT and Arf^−/− cells after treatment with 50 ng/mL IFNγ for 6 h. (G) Protein levels of iNOS were evaluated by immunoblotting after stimulation of peritoneal macrophages with 50 ng/mL IFNγ for 24 h. Results were obtained from three independent experiments performed in triplicate and data are reported as means ± SD *p < 0.05, **p < 0.01 and ***p < 0.001 as compared with the same condition in WT macrophages. (D) and (G) are representative of one out of three independent experiments.