1. A CDK-Independent Function of Mammalian Cks1
Charles Spruck, Heimo Strohmaier, Mark Watson, Adrian P.L Smith, Aimee Ryan, Wilhelm Krek, Steven I Reed 
Molecular Cell 
Volume 7, Issue 3, Pages (March 2001)
DOI: /S (01)

2. Figure 1 Targeted Disruption of the CKS1 Gene in Mice
(A) Schematic of the CKS1 locus, targeting construct pCKS1.KO and resultant mutant allele following homologous recombination. pCKS1.KO was designed to replace a 2.1 kb fragment of the CKS1 locus encoding exon 2 with a PGK-neo-poly(A) cassette. Filled boxes represent exons. Restriction sites include the following: H, HindIII; E, EcoRI; B, BamHI; X, XbaI. Striped box shows the probe used for Southern blot based genotyping. Arrows indicate PCR primers used in genotyping.
(B) Multiplex PCR based genotyping of F2 generation mice.
(C) Representative CKS1−/− and wild-type male mice depicting size difference.
(D) Growth curves for male and female CKS1−/−, CKS1+/−, and wild-type F2 generation littermates.
(E) Wild-type, heterozygous, and CKS1−/− littermate embryos at day 13.5
Molecular Cell 2001 7, DOI: ( /S (01) )

3. Figure 2 Reduced Proliferation of CKS1−/− MEFs
(A) Growth curves of wild-type (open squares) and CKS1−/− (filled circles) MEFs in medium containing various serum concentrations.
(B) Cell density at confluence. Wild-type (white) and CKS1−/− (gray) MEFS were grown for 10 days in medium containing 10% FBS and then counted
Molecular Cell 2001 7, DOI: ( /S (01) )

4. Figure 3
Loss of Cks1 Function Results in Accumulation of p27Kip1 and Cyclin E
(A) Immunoblot of various cell cycle regulatory proteins in asynchronously growing MEFs.
(B) Immunoblot of various proteins performed on CKS1−/−, heterozygous, and wild-type whole 13.5-day-old embryos.
(C) Levels of cell cycle regulatory proteins in CKS1−/− and wild-type MEFs during cell cycle progression. MEFs were arrested in G0/G1 by incubation in serum-free (0.1%) medium for 72 hr and stimulated to enter the cell cycle by readdition of serum (10%) to the medium.
(D) H1-kinase assays of CKS1−/− and wild-type MEFs following release from serum starvation.
(E) Immunoprecipitation of p27Kip1 and cyclin E from extracts of CKS1−/− and wild-type MEFs at various cell cycle phases immunoblotted for cyclin E, CDK2, p27Kip1, and p(Thr187)-p27Kip1
Molecular Cell 2001 7, DOI: ( /S (01) )

5. Figure 4
CKS1−/− Cells Are Deficient in Their Ability to Ubiquitinate p27Kip1
(A) Pulse–chase analysis of p27Kip1 in CKS1−/− and wild-type MEFs arrested in S phase of the cell cycle by the addition of 1.0 mM thymidine to the culture medium.
(B) In vitro ubiquitination assay of p27Kip1 using CKS1−/− and wild-type extracts. 35S-labeled mouse p27Kip1 was incubated with extract (120 μg) from asynchronously growing MEFs. The p27Kip1 substrate was phosphorylated by preincubation with CDK2/cyclin E where indicated. Recombinant human Cks1 was added to the reactions in lanes 6–8 and 11–14, and human Cks2 was added to the reaction in lane 9. The CDK2 inhibitor roscovitine (10–100 μM) was added to the extract and substrate phosphorylation reaction prior to the addition of Cks1 in lanes 13 and 14. Ubiquitin was supplemented to the reaction in lane 15, and increasing amounts of methylated ubiquitin were added to the reactions in lanes 16–19.
(C) Detection of in vivo p27Kip1 ubiquitin conjugates. Thymidine arrested MEFs were treated with MG132 (lanes 3 and 6) or DMSO (lanes 2 and 5) for 5 hr. Cell lysates were immunoprecipitated with anti-p27Kip1 antibodies (lanes 2, 3, 5, and 6) or with control IgG (lanes 1 and 4), separated by SDS–PAGE, blotted, and probed with anti-p27Kip1 antibodies. A darker exposure of the same blot, allowing detection of more slowly migrating forms of p27Kip1, is shown in the inset.
(D) p27Kip1 adenoviral transduction of wild-type MEFs arrested in S phase. Immunoblot of untransduced wild-type extract is shown in lane 1. Extract of wild-type MEFs transduced with increasing multiplicity of p27Kip1 adenovirus is shown in lanes 2–5. Lane 6 shows extract of wild-type MEFs infected with a control virus at multiplicity equal to the highest p27Kip1 adenovirus multiplicity. Untransduced CKS1−/− extract is shown in lane 7
Molecular Cell 2001 7, DOI: ( /S (01) )

6. Figure 5
Cks1 Mediates the Interaction between Skp2 and p27 In Vivo and Associates with Skp2
(A) Immunoprecipitation of Skp2 from CKS1−/− and wild-type extracts. Extracts from cells arrested in S phase were immunoprecipitated with anti-Skp2 antibodies and probed for Skp2 and p27Kip1 by immunoblotting. Rabbit nonimmune IgG was used in the control immunoprecipitations. Roscovitine (100 μM) was added to extract at 4°C for 1 hr prior to the addition of Cks1 protein in lane 7. Lanes 8 and 9 show the levels of p27Kip1 and Skp2 in thymidine-arrested extracts. H1 kinase reactions performed on CDK2 immunoprecipitates from parallel extracts are shown in lanes 11–14.
(B) Cks1 binds Skp2 in vitro. Various human Cks homologs were labeled with 35S-methionine and tested for their ability to bind GST-Skp2 bound to glutathione beads. GST-Ddi1 bound to glutathione beads was used a control in lane 4. The Cks1 (E63Q) mutant in lane 2 is defective in CDK binding. An excess of unlabeled recombinant Cks1 was added to the reaction in lane 6.
(C) TNFα-mediated degradation of IκB in wild-type and CKS1−/− MEFs. Asynchronously growing MEFs were treated with TNFα, and the cells were harvested at the indicated times. Lysates were immunoblotted with anti-IκB and anti-phospho-IκB antibodies.
(D) β-catenin levels in asynchonously growing wild-type and CKS1−/− MEFs. IκB levels are shown in the lower panel as a loading control
Molecular Cell 2001 7, DOI: ( /S (01) )

7. Figure 6
In Vitro Reconstitution of p27Kip1 Ubiquitination by SCFSkp2 Requires Cks1
(A) In vitro reconstitution of p27Kip1 ubiquitination activity using purified components. The CDK2/cyclin E, E3 core complex (Cul1, Skp1, Roc1), and SCFSkp2 (Cul1, Skp1, Roc1, Skp2) used were expressed from baculovirus and purified from Sf9 cells. Cks1, Cks1(E63Q) mutant, and Cks2 were expressed and purified from bacteria. The 35S-methionine-labeled p27Kip1 substrate was generated by an in vitro transcription/translation system. The reactions were supplemented with methylated-ubiquitin, ubiquitin, an ATP regenerating system, and E1 and Cdc34 enzymes. Roscovitine was added to the CDK2/cyclin E/p27Kip1 phosphorylation reaction prior to mixing with SCFSkp2.
(B) In vitro reconstitution of p27Kip1 ubiquitination using His-p27Kip1, expressed and purified from bacteria. Reactions were performed as described in (A). Ubiquitinated p27Kip1 was detected by immunoblotting and probing with anti-p27Kip1 antibodies.
(C) Association of Skp2/Skp1 with phosphorylated p27Kip1 contained in CDK2/cyclin E complexes. His-p27Kip1 was incubated with CDK2/cyclin E in the presence of 32[P-γ]-ATP. Cks1 and Cks1(E63Q) were incubated with Skp2/Skp1 dimer and then mixed with the p27Kip1 kinase reaction. Reactions were immunoprecipitated with anti-Skp2 antibodies and analyzed. Rabbit nonimmune IgG was used in the control lane
Molecular Cell 2001 7, DOI: ( /S (01) )

8. Figure 7
Model for Cks1 Regulation of p27Kip1 Ubiquitination by SCFSkp2
Cks1 binds to Skp2 and stimulates the targeting of phosphorylated p27Kip1 for ubiquitination by SCFSkp2. The binding of Cks1 to Skp2 is independent of its association with CDKs
Molecular Cell 2001 7, DOI: ( /S (01) )