Fig. 2. PP1 directly inhibits Kit in an IP-kinase assay. H526 cells were stimulated with SCF using cells pretreated with DMSO vehicle (Lanes A and C) or 10 µM PP1 (Lane B) for 30 min. A Kit IP was performed, followed by an in vitro kinase assay using radiolabeled ATP. PP1 (10 µM) was added to one reaction (Lane C) before the addition of ATP; an equivalent volume of DMSO was added to the others. The kinase reaction was electrophoretically resolved and transferred to nitrocellulose, and an autoradiograph was performed (upper panel), followed by staining for total Kit protein (lower panel). The addition of PP1 directly to the kinase reaction inhibited Kit autophosphorylation in vitro, whereas the addition of PP1 to cells prior to lysis had little effect on in vitro kinase activity.