Journal: Cell Death & Disease
Article Title: Pin1 inhibits PP2A-mediated Rb dephosphorylation in regulation of cell cycle and S-phase DNA damage
Figure Lengend Snippet: Pin1 inhibits Rb PP2a-mediated dephosphorylation. ( a ) WT or Pin1-deficient (Pin1 −/− ) MEF cell lysates were subjected to western blot analysis for total Rb levels (Pan-Rb), phosphorylated Rb (pRb) at Ser807/811 (S807/811), Pin1 or Actin. ( b ) H1299 cells were infected with recombinant retrovirus expressing shRNA against Pin1 or a vector control. Whole-cell lysates were subjected to western blotting, as shown. ( c ) MCF-10A cells were stably transfected with a PLVXpuro vector control (Vec) or PLVX-Pin1 (Pin1). Stable cells were treated with or without extraction buffer and subsequently subjected to immunofluorescence for Rb and counterstained with DAPI. ( d ) Rb C-pocket (RbC) peptides were in vitro phosphorylated and labeled with Cyclin E/CDK2 complexes with γ -[ 32 P]-ATP as described in the Materials and Methods. [ 32 P]-labeled phosphorylated RbC peptides were then incubated with BSA, okadeic acid or WT or mutant GST-Pin1 fusion proteins, before incubation with PP2A for the indicated times. Dephosphorylation reactions were quenched by the addition of SDS sample buffer. Samples were analyzed by SDS-PAGE followed by autoradiography
Article Snippet: In vitro phosphorylation and dephosphorylation First, 400 ng of RbC protein (Cell Signaling, #6022) were labeled by in vitro phosphorylation in a reaction system containing 40 ng Cyclin E/CDK2 (Cell Signaling, #7524), 30 μ Ci γ -[32 P]-ATP (NEN Life Sciences), 100 μ M ATP, 50 mM Tris-HCl pH 7.5, 10 mM MgCl2 , 1 mM EGTA, 2 mM DTT and 0.01% (w/v) Brij-35 by incubation at 30 °C for 30 min.
Techniques: De-Phosphorylation Assay, Western Blot, Infection, Recombinant, Expressing, shRNA, Plasmid Preparation, Stable Transfection, Transfection, Immunofluorescence, In Vitro, Labeling, Incubation, Mutagenesis, SDS Page, Autoradiography