THP-1 and U937 cells can rephosphorylate pRb after multiple days of PD0332991 treatment by reactivating CDK2. (A) Western blot. The indicated AML cell lines were treated with DMSO (vehicle control) or PD0332991 (500 nM) for 24 or 120 hours. Lysates were resolved by SDS-PAGE followed by immunoblotting with anti-pRb. (B) Western blot. Cells were treated for 24 or 96 hours with PD0332991 (500 nM) or DMSO (vehicle control). Lysates were subjected to SDS-PAGE followed by Western blot analysis with the indicated antibodies. An antibody against actin was used as a loading control. (C) CDK2 in vitro kinase assay: Cells were incubated for 96 hours with DMSO (vehicle control) or PD0332991 (500 nM). Cell lysates were prepared and normalized by protein concentration. CDK2 was immunoprecipitated to perform the in vitro kinase assay. Incorporation of ³²P in the CDK2 substrate histone H1 was assessed by SDS-PAGE followed by autoradiography. As a control for the kinase assay, a lysate from THP-1 cells was incubated with an isotype-matched antibody to form an immunocomplex. To confirm equal protein amounts in each normalized extract, aliquots were separated by SDS-PAGE and analyzed by Western blot with an anti-Grb-2 antibody. (D) Western blot and real-time PCR. The indicated AML cell lines were treated with DMSO (vehicle control) or PD0332991 (500 nM) for 96 hours. Lysates were prepared and resolved by SDS-PAGE followed by immunoblotting with phospho-Foxo3a (Thr32), Foxo3a, p27, and actin as a loading control. The cells were also used to prepare mRNA to determine by real-time PCR the pairwise changes in p27 mRNA levels between DMSO- and PD0332991-treated cells. Two primer pairs were used for the analysis.