TNFα-induced IRF-1 nuclear translocation. A, RASFs were preincubated for 2 hours with pyrrolidine dithiocarbamate (PDTC; 200 μM), PD98059 (10 μM), SB202190 (10 μM), or SP600125 (10 μM) and then stimulated for 2 hours with TNFα (20 ng/ml). IRF-1 nuclear translocation was determined by Western blotting, using the nuclear extract from RASFs. Bars show the mean ± SEM. B, RASFs were preincubated for 2 hours with PDTC (200 μM) or PD98059 (10 μM) and then stimulated with TNFα (20 ng/ml) for 2 hours. Localization of IRF-1 on RASFs with or without TNFα stimulation was examined using immunofluorescence and confocal microscopy. After 2 hours of stimulation with TNFα, IRF-1 (arrows) was expressed in the nucleus (left). After preincubation with PDTC, IRF-1 expression was more pronounced in the cytoplasm (middle), whereas preincubation with PD98059 did not affect the location of IRF-1 in the nucleus. The images are representative of 4 independent experiments. See Figure 1 for other definitions.