Figure 5.
Figure 5. G-CSF induced activation of p47phox. (A) Serine phosphorylation of p47phox induced by G-CSF. Ba/F3GR cells were treated with or without 100 ng/mL G-CSF for indicated time periods, and lysates were prepared. Proteins were immunoprecipitated with anti-p47phox antibody and blotted with either anti-phosphoserine antibody or anti-p47phox antibody. The latter demonstrates comparable protein loading. (B) Inhibition of serine phosphorylation of p47phox by kinase inhibitors. Ba/F3GR cells were untreated or pretreated with kinase inhibitors for 1 hour, followed by no or 100 ng/mL G-CSF for 60 minutes, then the lysates were prepared. Proteins were immunoprecipitated with anti-p47phox antibody and blotted with either anti-phosphoserine antibody or anti-p47phox antibody. (C) Membrane translocation of p47phox. Ba/F3GR cells were untreated or treated with 100 ng/mL G-CSF for 10 minutes, and then cells were lysed and subjected to ultracentrifugation. Membrane and cytosolic fractions were purified and then subjected to electrophoresis and immunoblotting with anti-p47phox antibody. Lane 1, whole-cell lysate from Ba/F3GR cells; lanes 2-3, whole-cell lysates after sonication; lane 4, cytosolic fraction of Ba/F3GR cells unstimulated by G-CSF; lane 5, cytosolic fraction of Ba/F3GR cells stimulated by G-CSF; lane 6, membrane fraction of Ba/F3GR cells unstimulated by G-CSF; lane 7, membrane fraction of Ba/F3GR cells stimulated by G-CSF. (D) Abrogation of membrane translocation of p47phox following treatment with Src or Akt kinase inhibitors. Ba/F3GR cells were pretreated with DMSO (-) or Src kinase inhibitor PP1 (P), the PI3K inhibitor Ly294 002 (L), the Akt inhibitor A838450 (A), or ERK1/2 inhibitor MEKI (M) for 1 hour, followed by stimulation with 100 ng/mL G-CSF for 10 minutes. Cytosolic and membrane fractions were prepared and analyzed for trafficking of p47phox. Comparable results were obtained in 3 independent experiments. (E) DPI, a specific inhibitor of NADPH oxidase, blocked G-CSF-induced ROS production. Ba/F3GR cells were pretreated with 0.1 μMor1 μM DPI for 1 hour, loaded with 5 μM DHR for 10 minutes, and then stimulated with 100 ng/mL G-CSF for 30 minutes. ROS production was determined by flow cytometry. Purple indicates untreated cells without G-CSF; green, untreated cells + G-CSF; pink, 0.1 μM DPI + G-CSF; blue, 1 μM of DPI + G-CSF.

G-CSF induced activation of p47phox. (A) Serine phosphorylation of p47phox induced by G-CSF. Ba/F3GR cells were treated with or without 100 ng/mL G-CSF for indicated time periods, and lysates were prepared. Proteins were immunoprecipitated with anti-p47phox antibody and blotted with either anti-phosphoserine antibody or anti-p47phox antibody. The latter demonstrates comparable protein loading. (B) Inhibition of serine phosphorylation of p47phox by kinase inhibitors. Ba/F3GR cells were untreated or pretreated with kinase inhibitors for 1 hour, followed by no or 100 ng/mL G-CSF for 60 minutes, then the lysates were prepared. Proteins were immunoprecipitated with anti-p47phox antibody and blotted with either anti-phosphoserine antibody or anti-p47phox antibody. (C) Membrane translocation of p47phox. Ba/F3GR cells were untreated or treated with 100 ng/mL G-CSF for 10 minutes, and then cells were lysed and subjected to ultracentrifugation. Membrane and cytosolic fractions were purified and then subjected to electrophoresis and immunoblotting with anti-p47phox antibody. Lane 1, whole-cell lysate from Ba/F3GR cells; lanes 2-3, whole-cell lysates after sonication; lane 4, cytosolic fraction of Ba/F3GR cells unstimulated by G-CSF; lane 5, cytosolic fraction of Ba/F3GR cells stimulated by G-CSF; lane 6, membrane fraction of Ba/F3GR cells unstimulated by G-CSF; lane 7, membrane fraction of Ba/F3GR cells stimulated by G-CSF. (D) Abrogation of membrane translocation of p47phox following treatment with Src or Akt kinase inhibitors. Ba/F3GR cells were pretreated with DMSO (-) or Src kinase inhibitor PP1 (P), the PI3K inhibitor Ly294 002 (L), the Akt inhibitor A838450 (A), or ERK1/2 inhibitor MEKI (M) for 1 hour, followed by stimulation with 100 ng/mL G-CSF for 10 minutes. Cytosolic and membrane fractions were prepared and analyzed for trafficking of p47phox. Comparable results were obtained in 3 independent experiments. (E) DPI, a specific inhibitor of NADPH oxidase, blocked G-CSF-induced ROS production. Ba/F3GR cells were pretreated with 0.1 μMor1 μM DPI for 1 hour, loaded with 5 μM DHR for 10 minutes, and then stimulated with 100 ng/mL G-CSF for 30 minutes. ROS production was determined by flow cytometry. Purple indicates untreated cells without G-CSF; green, untreated cells + G-CSF; pink, 0.1 μM DPI + G-CSF; blue, 1 μM of DPI + G-CSF.

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