Figure 2.

Effect of specific inhibitors of Janus kinase 2 (JAK2), extracellular signal-regulated kinase 1 and 2 (Erk1/Erk2) and MAPK kinase 1 and 2 (MEK1/2) on interferon-γ (IFN-γ)-induced macrophage nitric oxide (NO) generation, and inducible nitric oxide synthase (iNOS) gene and protein expression. Cells were treated with increasing doses of AG-490 (JAK2 inhibitor) (a), apigenin (Erk1/Erk2 inhibitor) (b) or PD 98059 (MEK1/2 inhibitor) (c) for 1 hr prior to stimulation with IFN-γ (100 U/ml). After 24 hr of incubation, supernatants were collected and submitted to the Greiss reaction to evaluate nitrite generation (mean ± SEM, n = 3). (d) to (f) iNOS regulation has been monitored in cells pretreated, as described above, with specific inhibitors prior to stimulation with IFN-γ for 8 hr (iNOS gene expression) or 24 hr (iNOS protein expression). After incubation, either total RNA or total proteins were extracted, and used for Northern and Western blots, respectively. These results are representative of one of three experiments performed independently. GAPDH, glyceraldehyde 3-phosphate dehydrogenase probe used to confirm equal RNA loading.