Fig. 2.
Fig. 2. cAMP cooperates with CSF-1 to synergistically activate ERK/MAPK. 32D/CSF-1R cells were starved in serum-free media for 2 hours. Where indicated, 50 μmol/L forskolin (forsk) and 0.5 mmol/L IBMX or 1 mmol/L btcAMP were added during the last 20 minutes of the incubation or as indicated and then stimulated with or without CSF-1 for 4 minutes or as indicated at 37°C. Immune complex kinase assays were performed on lysates normalized for protein content. Fold refers to increase in substrate phosphorylation relative to that in untreated cells. (A) ERK2 was immunoprecipitated and activity measured in an in vitro kinase assay with MBP as a substrate. The left panel shows that similar results were obtained with either a 10- or 30-minute pretreatment period with forskolin/IBMX and with btcAMP. The right panel shows that forskolin/IBMX pretreatment increased ERK activity at both 1 and 4 minutes after CSF-1 addition. The rightmost panel is a longer exposure to compare basal ERK activity with that in the presence of forskolin/IBMX only. The magnitude of the increase induced by forskolin/IBMX varies between experiments (see Fig 3). (B) Immunoprecipitated ERK2 was analyzed for tyrosine phosphorylation by Western blotting with an antiphosphotyrosine antibody (top). The blot was then stripped and reprobed with anti-ERK2 monoclonal antibody (bottom). (C) Cells were pretreated with varying doses of forskolin or IBMX as indicated, followed by CSF-1 stimulation. ERK immune complex kinase assays were performed as described above. (D) Cells were stimulated with CSF-1 alone (▪), pretreated with forskolin/IBMX for 20 minutes and then stimulated with CSF-1 (•), or treated with CSF-1 and forskolin/IBMX simultaneously (▵). Aliquots were taken at the indicated time points, and lysates were prepared and assayed for ERK2 activity. (E) MEK1 was immunoprecipitated and activity measured with recombinant KD MAPK protein as substrate (left panel). MEK1 or MEK2 was immunoprecipitated from the same preparation of lysates and assayed for kinase activity (right panel). Parallel MEK1 and MEK2 immune complex kinase assays were repeated twice with similar results.

cAMP cooperates with CSF-1 to synergistically activate ERK/MAPK. 32D/CSF-1R cells were starved in serum-free media for 2 hours. Where indicated, 50 μmol/L forskolin (forsk) and 0.5 mmol/L IBMX or 1 mmol/L btcAMP were added during the last 20 minutes of the incubation or as indicated and then stimulated with or without CSF-1 for 4 minutes or as indicated at 37°C. Immune complex kinase assays were performed on lysates normalized for protein content. Fold refers to increase in substrate phosphorylation relative to that in untreated cells. (A) ERK2 was immunoprecipitated and activity measured in an in vitro kinase assay with MBP as a substrate. The left panel shows that similar results were obtained with either a 10- or 30-minute pretreatment period with forskolin/IBMX and with btcAMP. The right panel shows that forskolin/IBMX pretreatment increased ERK activity at both 1 and 4 minutes after CSF-1 addition. The rightmost panel is a longer exposure to compare basal ERK activity with that in the presence of forskolin/IBMX only. The magnitude of the increase induced by forskolin/IBMX varies between experiments (see Fig 3). (B) Immunoprecipitated ERK2 was analyzed for tyrosine phosphorylation by Western blotting with an antiphosphotyrosine antibody (top). The blot was then stripped and reprobed with anti-ERK2 monoclonal antibody (bottom). (C) Cells were pretreated with varying doses of forskolin or IBMX as indicated, followed by CSF-1 stimulation. ERK immune complex kinase assays were performed as described above. (D) Cells were stimulated with CSF-1 alone (▪), pretreated with forskolin/IBMX for 20 minutes and then stimulated with CSF-1 (•), or treated with CSF-1 and forskolin/IBMX simultaneously (▵). Aliquots were taken at the indicated time points, and lysates were prepared and assayed for ERK2 activity. (E) MEK1 was immunoprecipitated and activity measured with recombinant KD MAPK protein as substrate (left panel). MEK1 or MEK2 was immunoprecipitated from the same preparation of lysates and assayed for kinase activity (right panel). Parallel MEK1 and MEK2 immune complex kinase assays were repeated twice with similar results.

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