cAMP synergizes with oncogenic Ras to stimulate ERK in an MEK1-dependent manner. 32D/CSF-1R cells were cotransfected with vector (7 μg) and HA-ERK2 (2 μg) or with 61LRas (6 μg), HA-ERK2 (1 μg), and vector (2 μg). Twenty-four hours later, surviving cells were starved and pretreated or not with forskolin/IBMX before stimulation with CSF-1 for 4 minutes. (A) Transfected ERK2 from lysates normalized to contain equivalent levels of HA-ERK2 was immunoprecipitated with anti-HA antibody and kinase activity towards MBP was determined (top). Anti-HA Western blot of transfected HA-ERK2 is shown (bottom). The results are representative of four independent sets of transfections. (B) Cells were cotransfected with 61LRas (3 μg), HA-ERK2 (1 μg), and the indicated amounts of dn-MEK1, with the total DNA content kept constant with empty vector. HA-ERK2 kinase activity (top) and Western blot (bottom) are shown. (C) FACS analysis shows Ras expression level in cells 24 hours after transfection with either vector (20 μg) or 6LRas (20 μg). Cells were permeabilized and stained with anti-Ras antibody (Y13-258). As a negative control, Ras-transfected cells were stained with an irrelevant primary antibody (anti-CD4). Abscissa: log relative fluorescence intensity; ordinate: relative cell number. Raf-1 kinase activity in cells transfected with 61LRas, in the absence or presence of cAMP-elevating agents, is shown on the right.