(a) Immunoblot analysis of HeLa cells treated with 20μM of the PI 3-K inhibitor LY294002 for the indicated duration of time.
(b–c) Immunoblot analysis of HeLa cells transfected with the indicated siRNA oligonucleotides. The control lane is scrambled E2F-1 siRNA; Luciferase, siRNA against firefly luciferase; PTEN1–3; three independent PTEN siRNA oligos; siRNA, short interfering RNA.
(d) Immunoblot analysis of HeLa cells transfected with the indicated siRNA oligos, after synchronization with nocodazole and release.
(e) Immunoblot analysis of indicated cell lines cultured in serum-free medium. Whole cell lysates were isolated in the presence of phosphatase inhibitors.
Full-length blots are provided in the Supplementary Information, Fig. S11.

(a) Sequence alignment of the putative Akt phosphorylation site at S72 in Skp2 from different species.
(b) Immunoblot (IB) analysis of whole cell lysates (WCL) and immunoprecipitates (IP) derived from 293T cells transfected with HA.Myr.Akt and FLAG.Skp2 constructs.
(c–d) Immunoblot (IB) analysis of whole cell lysates (WCL) and immunoprecipitates (IP) derived from 293T cells transfected with HA.Skp2 or empty vector (EV). Cdk2 and Cylin A antibodies were used as positive controls to detect the interaction with Skp2, while the LSD1 antibody was used as a negative control.
(e) Immunoblot (IB) analysis of whole cell lysates (WCL) and immunoprecipitates (IP) derived from 293T cells transfected with indicated HA.Myr.Akt1 or HA-Myr.Akt2 and FLAG.Skp2 constructs.
(f) Autoradiography of ³⁵S-labelled Akt1 or Akt2 bound to the indicated GST-fusion proteins.
(g) Immunoblot (IB) analysis of whole cell lysates (WCL) and immunoprecipitates (IP) derived from 293T cells transfected with indicated HA.Myr.Akt1 and FLAG.Skp2 constructs.
(h) Immunoblot (IB) analysis of whole cell lysates (WCL) and immunoprecipitates (IP) derived from 293T cells transfected with indicated FLAG.Skp2 construct or empty vector (EV). Cdk2 antibody was used as a positive control to detect the interaction with Skp2.
Full-length blots are provided in the Supplementary Information, Fig. S11.

(a) In vivo Skp2 phosphorylation sites detected by mass spectrum analysis.
(b) Immunoblot (IB) analysis of whole cell lysates (WCL) and immunoprecipitates (IP) derived from 293T cells transfected with HA.Skp2. The Cylin A antibody was used as a positive control to detect the interaction with Skp2, while the TSC1 antibody was used as a negative control.
(c) Immunoblot analysis of HeLa and U2OS cells treated with the Casein Kinase I inhibitors D4476 and IC261 at the indicated concentrations for 12 hours.
(d) Protein sequence illustration of the phosphorylation of the Ser75 site by Casein Kinase I (CKI), which is triggered by the phosphorylation of the Ser72 site by Akt1. The same sequential phosphorylation cascade has been described in the Foxo family of transcription factors.
(e–f) CKI phosphorylates Skp2 in vitro at S75. Purified CKI protein (from New England Biolabs) was incubated with 5 μg of indicated GST.Skp2 in the presence of γ-³²P-ATP. The kinase reaction products were resolved by SDS-PAGE and phosphorylation was detected by autoradiography.
Full-length blots are provided in the Supplementary Information, Fig. S11.

(a) HeLa cells were transfected with the indicated FLAG.Skp2 plasmids. 20 hr post-transfection, cells were split into 60 mm dishes, and after another 20 hr, treated with 20 μg/ml cycloheximide. At the indicated time points, whole-cell lysates were prepared and immunoblots were probed with the indicated antibodies.
(b) Quantification of the band intensities in a. Skp2 band intensity was normalized to GFP, then normalized to the t=0 controls. Three independent sets of experiments were performed to generate the error bar. The error bars represent s.d.
(c) Immunoblot analysis of HeLa cells transfected with limiting amounts of FLAG.Skp2 (wild-type; S72A; S72D/S75D) plasmids with or without HA.Myr.Akt, along with a green fluorescent protein (GFP) as transfection control. HeLa cells were synchronized in M phase with nocodazole, and then released to the G1 phase for the indicated time periods.
(d) Quantitation of the band intensities in c. Skp2 band intensity was normalized to Tubulin, then normalized to the t=0 control of wild-type Skp2.
(e) Immunoblot analysis of whole cell lysates (WCL) and immunoprecipitates (IP) derived from HeLa cells transfected with the FLAG.Skp2 construct together with the indicated siRNA oligos.
(f) Immunoblot analysis of whole cell lysates (WCL) and immunoprecipitates (IP) derived from 293T cells transfected with the indicated FLAG.Skp2 constructs.
(g) Immunoblot analysis of whole cell lysates (WCL) and immunoprecipitates (IP) derived from HeLa cells transfected with the indicated FLAG.Skp2 constructs in the presence or absence of HA.Myr.Akt.
Full-length blots are provided in the Supplementary Information, Fig. S11.

(a) Real-time RT-PCR analysis to examine the relative Skp2 mRNA expression levels in HeLa cells transfected with the indicated siRNA oligonucleotides. Three independent sets of experiments were performed to generate the error bar. The error bars represent s.d.
(b) Immunoblot analysis to examine the relative Skp2 protein levels in HeLa cells transfected with the indicated siRNA oligonucleotides. Three independent sets of experiments were performed to generate the error bar. The error bars represent s.d.
(c) HeLa cells were transfected with indicated siRNA oligos. 40 hr post-transfection, cells were treated with 20 μg/ml cycloheximide. At the indicated time points, whole-cell lysates were prepared and immunoblots were probed with indicated antibodies.
(d) Quantification of the band intensities in c. Skp2 band intensity was normalized to Tubulin, then normalized to the t=0 controls. Three independent sets of experiments were performed to generate the error bar. The error bars represent s.d.
Full-length blots are provided in the Supplementary Information, Fig. S11.

(a) Akt phosphorylates Skp2 in vitro at S72. HA.Myr.Akt was transfected into 293T cells, recovered by anti-HA immunoprecipitation and incubated with 5 μg of indicated GST.Skp2 in the presence of γ-³²P-ATP. The kinase reaction products were resolved by SDS-PAGE and phosphorylation was detected by autoradiography.
(b) Akt phosphorylates Skp2 in vitro at S72. HA.Myr.Akt was transfected into 293T cells, recovered by anti-HA immunoprecipitation and incubated with 5 μg of indicated GST.Skp2 in the presence of cold ATP. The kinase reaction products were resolved by SDS-PAGE and phosphorylation was detected by the phospho-Akt-substrate antibody that recognizes either the RXRXXpS/pT or the R/KXR/KXXpS/pT motif.
(c) Immunoblot (IB) analysis of whole-cell lysates (WCL) and immunoprecipitates (IP) derived from HeLa cells transfected with HA.Myr.Akt and FLAG.Skp2. WT, wild-type Skp2; S72A, mutant Skp2 at the Akt site.
(d) Immunoblot (IB) analysis of whole-cell lysates (WCL) and immunoprecipitates (IP) derived from 293T cells transfected with HA.Myr.Akt and indicated FLAG.Skp2 plasmids. Where indicated, the whole cell lysates were treated with λ-phosphatase prior to immunoprecipitation.
(e) Immunoblot analysis of whole cell lysates (WCL) and immunoprecipitates (IP) derived from HeLa cells infected with Akt1 and Akt2 lentiviral shRNA. Endogenous Skp2 was immunoprecipitated with anti-Skp2 and immunoblotted with the Akt substrate directed phospho-antibody.
(f) Immunoblot analysis of whole cell lysates (WCL) and immunoprecipitates (IP) derived from HeLa cells transfected with the indicated kinases and FLAG.Skp2 construct.
(g) Mouse Skp2 protein is a poor Akt substrate. HA.Myr.Akt was transfected into 293T cells, recovered by anti-HA immunoprecipitation and incubated with 5 μg of indicated GST.Skp2 in the presence of γ-³²P-ATP. The kinase reaction products were resolved by SDS-PAGE and phosphorylation was detected by autoradiography.
Full-length blots are provided in the Supplementary Information, Fig. S11.

(a) Autoradiography of ³⁵S-labelled Cdh1 bound to the indicated GST-fusion proteins.
(b) Immunoblot analysis of whole cell lysates (WCL) and immunoprecipitates (IP) derived from 293T cells transfected with the FLAG.Skp2 construct in the presence or absence of HA.Myr.Akt.
(c) Immunobot analysis of HeLa cells transfected with the indicated FLAG.Skp2 and HA.Cdh1 plasmids in the presence or absence of HA.Myr.Akt. A plasmid encoding GFP was used as a negative control for transfection efficiency.
(d) Immunoblot analysis of HeLa cells transfected with the indicated FLAG.Skp2 plasmids and siRNA oligos. A plasmid encoding GFP was included as negative control for transfection efficiency.
(e) Immunoblot analysis of HeLa cells transfected with the indicated siRNA oligos, synchronized by growth in nocodazole, and then released for the indicated periods of time.

(a) Sequence alignment of Skp2 with p27, p21 and FOXO1 Nuclear Localization Sequence.
(b) Immunofluorescence and DAPI staining of HeLa cells transfected with FLAG-wild-type or ΔNLS Skp2 plasmids. Scale bars are 20 μm.
(c) Immunofluorescence and DAPI staining of HeLa cells transfected with indicated FLAG.Skp2 in the presence or absence of HA.Myr.Akt constructs. Scale bars are 20 μm.
(d) Immunofluorescence and DAPI staining of SKBR3 cells treated with LY294002 (or DMSO as negative control) for 12 hours. Scale bars are 20 μm.
(e) Immunoblot analysis of Nuclear (N) and Cytoplasmic (C) fraction of HeLa, MDA-MB468 and SKBR3 cells treated with LY294002 (or DMSO as a negative control) for 12 hours.
(f–h) Autoradiography of ³⁵S-labelled importin α1 (f), importin α5 (g) and importin α7 (h) bound to the indicated GST-fusion proteins.