(A) His-p27WT or His-p27Y74F were reacted with Src in vitro. p27 precipitates were blotted with αpY-4G10 or αp27pY74 (left). MCF-7 was transfected with YFPp27WT and empty vector (Src −) or PCI-Src-Y530F (Src +). p27 precipitates were blotted with αpY-4G10, stripped and re-probed for p27 (middle panel). pY74p27 detected in Src transfected MCF-7 (right panel).
(B) MCF-7 was co-transfected with YFPp27WT or mutant p27 with or without Src. pYp27 was detected in p27 precipitates with αpY-4G10 or αp27pY74.
(C) Cyclin E, Cdk2 and YFPp27 levels in YFPp27WT transfected MCF-7 with or without Src cotransfection or PP1.
p27 (D) and Cdk2 (E) precipitates were resolved and proteins detected by blotting.