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.