FIG. 8.
Effects of MEK variants in HEK293T cells. HEK293T cells were transiently transfected with MEK variants, serum deprived, and then harvested in the absence of additional stimulation. Extracts were probed with anti-phospho p44MAPK1 and -p42MAPK2 antibodies as described in Materials and Methods. Cells were treated with 1 μM PD 184352 for 1 h prior to harvesting. FLAG-tagged MEK expression and total p44MAPK1/p42MAPK2 levels were assessed by immunoblotting with anti-FLAG and anti-p44 MAPK1/p42MAPK2 antibodies, respectively. After treatment with 1 μM PD 184352, the Leu115Pro variant retained greater than 95% activity, as determined by densitometry quantitation (Kodak Imaging Systems) of a phospho-p44MAPK1/p42MAPK2 Western blot. The results are representative of three independent experiments.