American Society of Hematology

Figure 1

$Figure 1. Effects of IFN-γ on MAPK activation. Bone marrow–derived macrophages were obtained after 7 days of culture in the presence of M-CSF. The cells were rendered quiescent by incubating them in medium supplemented with 10% FCS (without M-CSF) for 15 hours before the start of the experiment. In panels A through C, G and H, quiescent macrophages were first treated with IFN-γ (10 ng/mL) or vehicle for 5 minutes and then stimulated with M-CSF (30 ng/mL) for the indicated periods of time. In panels D through F, quiescent macrophages were treated with IFN-γ (10 ng/mL) for the indicated periods of time. (A) The activity of ERK-1 and -2 was analyzed by in-gel kinase assay. As a control for sample quality and quantity, 20 μg whole-cell extract was used to measure β-actin expression by Western blotting. (B) JNK-1 activation was determined by in vitro kinase assay using c-Jun as a substrate. As a control for sample quality and quantity, 20 μg whole-cell extract was used to measure β-actin expression by Western blotting. (C) p38 activation was measured by Western blotting using an anti–phospho-p38 antibody. The expression of β-actin was measured as a control. (D) The phosphorylation of ERK-1 and -2 was measured by Western blotting, using anti–phospho-ERK antibodies. The expression of β-actin was measured as a control. (E) JNK-1 activation was determined by in vitro kinase assay. The loading control for section E is the same than in section D. (F) p38 activation was measured by Western blotting using anti–phospho-p38 antibodies. (G) Confocal microscopy was used to visualize translocation of total ERK to the nucleus. (H) Cytosolic and nuclear fractions were obtained from cells incubated with M-CSF or IFN-γ + M-CSF for the indicated periods of time. The samples were immunoblotted using antibodies specific against diphosphorylated ERK and total ERK. As a control, the expression of nuclear histone H1 was also analyzed. The experiments in panels A and G have been performed 5 times. The rest of the sections have been reproduced 3 times.$

Effects of IFN-γ on MAPK activation. Bone marrow–derived macrophages were obtained after 7 days of culture in the presence of M-CSF. The cells were rendered quiescent by incubating them in medium supplemented with 10% FCS (without M-CSF) for 15 hours before the start of the experiment. In panels A through C, G and H, quiescent macrophages were first treated with IFN-γ (10 ng/mL) or vehicle for 5 minutes and then stimulated with M-CSF (30 ng/mL) for the indicated periods of time. In panels D through F, quiescent macrophages were treated with IFN-γ (10 ng/mL) for the indicated periods of time. (A) The activity of ERK-1 and -2 was analyzed by in-gel kinase assay. As a control for sample quality and quantity, 20 μg whole-cell extract was used to measure β-actin expression by Western blotting. (B) JNK-1 activation was determined by in vitro kinase assay using c-Jun as a substrate. As a control for sample quality and quantity, 20 μg whole-cell extract was used to measure β-actin expression by Western blotting. (C) p38 activation was measured by Western blotting using an anti–phospho-p38 antibody. The expression of β-actin was measured as a control. (D) The phosphorylation of ERK-1 and -2 was measured by Western blotting, using anti–phospho-ERK antibodies. The expression of β-actin was measured as a control. (E) JNK-1 activation was determined by in vitro kinase assay. The loading control for section E is the same than in section D. (F) p38 activation was measured by Western blotting using anti–phospho-p38 antibodies. (G) Confocal microscopy was used to visualize translocation of total ERK to the nucleus. (H) Cytosolic and nuclear fractions were obtained from cells incubated with M-CSF or IFN-γ + M-CSF for the indicated periods of time. The samples were immunoblotted using antibodies specific against diphosphorylated ERK and total ERK. As a control, the expression of nuclear histone H1 was also analyzed. The experiments in panels A and G have been performed 5 times. The rest of the sections have been reproduced 3 times.

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