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. 2000 Sep;20(18):6779–6798. doi: 10.1128/mcb.20.18.6779-6798.2000

FIG. 1.

FIG. 1

PI3-kinase inhibitors block CSF-1-stimulated MEK/ERK but not Raf-1 activities. (A) ERK activity. 32D cells expressing the WT CSF-1R were starved and then pretreated or not with 300 nM wortmannin (Wort) or 50 μM LY294002 (LY) for 20 min before stimulation with 10 nM CSF-1 for 4 min. Lysates were immunoprecipitated (IP) with anti-ERK2 antibodies and subjected to an IVK reaction with MBP as substrate. The right panel shows the results of a dose-dependent experiment with the indicated amounts of wortmannin. (B) PI3-kinase activity. Cells were starved, not treated, or pretreated with 300 nM wortmannin before CSF-1 stimulation for the indicated times. PI3-kinase activity was assayed in anti-PY (4G10) immunoprecipitates with PI as substrate. Origin and PIP indicate the loading position and migration of a PI-4-P standard. (C) Effect of PI3-kinase inhibition on ERK phosphorylation at different CSF-1 concentrations. Cells were starved and pretreated or not with 200 nM wortmannin or 30 μM LY294002 before stimulation with the indicated CSF-1 concentrations. Total cell lysates were immunoblotted (IB) with an antibody that recognized only dually phosphorylated ERK. (D) MEK and Raf-1 activities. Cells were treated as described for panel A, and a MEK1 (left panel) or Raf-1 (right panel) immune complex kinase assay was performed with KD-MAPK or KD-MEK, respectively, as substrate. (E) Summary of results from the indicated number (n) of experiments showing the means ± standard errors. Data for ERK and MEK were averaged from experiments using 100 or 300 nM wortmannin as indicated or 50 μM LY294002; data for Raf-1 were averaged from experiments using 300 nM wortmannin or 50 μM LY294002. Data are expressed as the percentages of CSF-1-mediated increase in kinase activity over unstimulated cells, where 100% denotes the activation in the presence of CSF-1 only. Statistically significant differences between untreated and wortmannin or LY-treated samples are denoted by asterisks: ∗, P < 0.05; ∗∗, P < 0.005 (Student's two-sided t test).