Phosphorylation of proto-oncogene Raf-1 and activation of MEK in Mo7e, Meg-01, and Dami/HEL cells. (A) Phosphorylation of Raf-1 was examined by band shift on Western blot. Equal amounts of cellular proteins (40 μg) from Mo7e, Dami/HEL, Meg-01, and U937 cells incubated with no cytokine (lanes marked as C) or 40 ng/mL GM-CSF (lanes, GM) were subjected directly to 10.5% SDS-PAGE. The separated proteins were transferred to a PVDF membrane and probed with anti–Raf-1 antibody. The arrow indicates the location of unphosphorylated Raf-1 and the arrowhead indicates the band shift of phosphorylated Raf-1. (B) Activation of MEK was detected by the kinase-induced phosphorylation of a kinase-defective p42^mapk (K52R) labeled with [³²P]-ATP. Equal amounts of cellular proteins (30 μg) from Mo7e, Meg-01, and Dami/HEL cells incubated with no cytokine (lanes marked as C) or 40 ng/mL GM-CSF (lanes, GM) were used to detect MEK activity. Phosphorylation of p42 is used as the readout to detect MEK activity.