MEK activity is required for the synergistic activation of ERK by CSF-1 and cAMP. (A) 32D/CSF-1R cells were cotransfected with HA-ERK2 (2 μg) and either vector (10 μg) or dn-MEK1 (10 μg). Cells were processed 24 hours later as described in Fig 5. Western blot of total MEK1 (endogenous plus transfected) is shown in the top panel. MBP kinase activity in HA-ERK2 immunoprecipitates was determined and the results from four independent cotransfections with HA-ERK2 and dn-MEK1 or vector control are shown in the bottom panel (means ± SE). To emphasize the effect of dn-MEK1, HA-ERK activities measured in the presence of dn-MEK1 are normalized to those from vector-control cells for each condition. (B) 32D/CSF-1R cells were starved and treated with 100 μmol/L PD98059 for 1 hour and 40 minutes before the addition of forskolin/IBMX. This was followed 20 minutes later by CSF-1 or IL-3. Endogenous ERK2 was immunoprecipitated and MBP kinase activity determined. The experiment has been repeated with 50 μmol/L PD98059 with very similar results.