Fig. 3. PP1 blocks both SCF-mediated Kit and MAPK activation. In A, H526 cells incubated in serum-free medium overnight were either left unstimulated or stimulated for 5 min with 100 ng/ml of SCF after a 30-min pretreatment with the indicated concentrations of PP1. Kit was immunoprecipitated and the immunoprecipitate was immunoblotted with antiphosphotyrosine and anti-Kit antibodies in succession. The degree of Kit activation was calculated by densitometry (ratio of pTyr:Kit signal) and normalized to the SCF-stimulated vehicle control lane which was arbitrarily assigned a value of 100%. Experiment shown is representative of three replicates. In B, cells were treated as above, but after SCF treatment, whole-cell lysates were made in SDS buffer. The lysates were immunoblotted and stained successively with antiphospho-ERK and antipan-ERK antibodies. The degree of activation was calculated as above. Experiment shown is representative of three replicates.