1. Volume 11, Issue 5, Pages 1189-1200 (May 2003)
The F-Box Protein Skp2 Participates in c-Myc Proteosomal Degradation and Acts as a Cofactor for c-Myc-Regulated Transcription 
Natalie von der Lehr, Sara Johansson, Siqin Wu, Fuad Bahram, Alina Castell, Cihan Cetinkaya, Per Hydbring, Ingrid Weidung, Keiko Nakayama, Keiichi I Nakayama, Ola Söderberg, Tom K Kerppola, Lars-Gunnar Larsson 
Molecular Cell 
Volume 11, Issue 5, Pages (May 2003)
DOI: /S (03)00193-X

2. Figure 1 c-Myc Binds to Skp2 In Vivo and In Vitro
(A) Coimmunoprecipitation (CoIP) of c-Myc and Cul1. Flag-tagged c-Myc and HA-tagged Cul1 expression vectors were transfected into Cos 7 cells. α-FLAG and α-HA sera were used for immunoprecipitations (IP) of Myc and Cul1, respectively, while α-Myc and α-HA sera were used for Western (W) analysis.
(B and C) CoIP of Skp2 and c-Myc. Cos 7 cells transfected with the indicated vectors were treated with 50 μM MG115 for 2 hr prior to lysis. IP and W were performed as indicated.
(D) CoIP of endogenous Myc and Skp2. HeLa cells were lysed in low-stringency buffer, and endogenous proteins were analyzed by IP and W as indicated.
(E) Direct association of Myc and Skp2 in vitro. GST pull-downs were performed using GST fusion proteins and IVT proteins as indicated (upper panel). The input of GST fusion protein was analyzed by W (lower panel).
Molecular Cell  , DOI: ( /S (03)00193-X)

3. Figure 2
The c-Myc/Skp2 Interaction Requires MB2 and the HLH-Zip Domain of c-Myc and Occurs in the Nucleus
(A) Structure of c-Myc and overview of Flag-tagged c-Myc deletion derivatives.
(B) CoIP analysis of interactions between Skp2 and c-Myc deletion derivatives. wt c-Myc and mutants were cotransfected with Skp2 into Cos 7 cells. Cells were treated with MG115 as in Figure 1B.
(C) GST pull-down analysis of Skp2 interactions with c-Myc deletion derivatives in vitro. GST pull-downs were performed using GST fusion proteins and IVT products as indicated. Input of 35S IVT protein is shown.
(D) Bimolecular fluorescence complementation (BiFC) analysis of Myc-Skp2 interactions in living cells. Cos 7 cells were transfected with YN and YC fusion constructs as indicated, and the cells were analyzed by fluorescence microscopy. Myc-EGFP was used as reference. DAPI staining was used to visualize cell nuclei.
Molecular Cell  , DOI: ( /S (03)00193-X)

4. Figure 3 Skp2 Regulates c-Myc Proteasome-Mediated Turnover
(A) Skp2 stimulates c-Myc turnover. HeLa cells were cotransfected using the expression vectors indicated together with Cul1, and the cells were treated with CHX 36 hr after transfection to block protein synthesis. Cell lysates prepared at the indicated times were immunoprecipitated using α-Flag antibody to precipitate tagged c-Myc and immunoblotted using the same antibody (upper panels). Quantitation of c-Myc turnover following CHX chase based on densitometrical scanning of experiments is shown (lower panel).
(B) The dominant-negative Skp2ΔF mutant stabilizes c-Myc. HeLa cells were transfected using the expression vectors indicated, and CHX treatment and Myc analysis were performed as in (A). Lower panel: quantitation of c-Myc levels following CHX treatment by densitometrical scanning of autoradiograms.
(C) Titration of Skp2. HeLa cells were transfected with 0.33 μg c-Myc, 1 μg Skp2ΔF, and increasing amounts (0.1–1 μg) Skp2 wt and steady-state levels of c-Myc were analyzed by α-Flag IP and Western.
(D) Effects of siRNA treatment on c-Myc degradation. HeLa cells were treated with siRNA-oligos specific for Skp2 or luciferase (Luc), or were left untreated (–) for 72 hr. Skp2, c-Myc, and p27 levels were analyzed using the antibodies indicated (upper left panels). Lower left panel: levels of c-Myc following CHX treatment for indicated times as described in (B). Right panel: quantitation of c-Myc turnover following siRNA as above or after lovastatin (LS), hydroxyurea (HU), or aphidicolin (AC) treatment.
(E) Effects of mutations in c-Myc on its stability. c-Myc turnover was analyzed in HeLa cells transfected with the indicated Myc constructs followed by CHX treatment as in (B). The graphs in (A)–(E) are based on data from at least three experiments.
Molecular Cell  , DOI: ( /S (03)00193-X)

5. Figure 4
c-Myc Ubiquitylation Is Reduced by Dominant-Negative Skp2 and by Mutations that Reduce Skp2 Binding
(A) U2OS cells were transfected using the expression vectors indicated, and the cells were treated with MG115. His-Ub-conjugated proteins were purified by Co2+ agarose, and the captured proteins were analyzed by α-Flag immunoblot (upper panel) or by dot blot analysis using α-His (lower left) or α-HA (lower right) sera. The right outermost dot represents lysate from untransfected control cells. The middle panel shows the c-Myc levels in 1/5 of the input material.
(B) U2OS cells were transfected as indicated using constant amounts of c-Myc, increasing amounts of Skp2ΔF (lanes 3–5), and increasing amounts of Skp2wt (lanes 6–8). Analysis of His-Ub bound proteins and total lysate was performed as in (A).
(C) U2OS cells were transfected using the indicated constructs, and extracts were analyzed as in (A) using Ni2+-agarose.
Molecular Cell  , DOI: ( /S (03)00193-X)

6. Figure 5
Skp2 and c-Myc Interact during the G1 to S Phase Transition of the Cell Cycle
(A) Endogenous c-Myc/Skp2 interactions in human PBLs activated with PHA and IL-2 for the indicated times (upper panel). Expression of c-Myc, Skp2, p27, and cyclin A was analyzed as indicated. Ponceau staining was used to normalize loading.
(B) Northern blot analysis of c-myc and gapdh mRNA levels in activated PBLs.
(C) Analysis of c-Myc protein synthesis in activated PBLs by 35S pulse labeling followed by IP. An equal amount of TCA-precipitable counts was used.
(D and E) Skp2 promotes c-Myc-induced S phase transition in Rat1 and p27−/− MEF cells. (D) Rat1MycER cells transfected with Skp2 or Skp2ΔF together with EGFP were FACS sorted and serum starved for 48 hr in the presence or absence of 4-OHT, after which the cell cycle distribution was analyzed by FACS. The results are presented as the ratio between the percentage of cells in S and G1. S/G1 ratio for exponentially growing cells (exp. cells) is shown as reference. (E) The same experiments as in (D) were performed using p27−/−MEF MycER cells.
Molecular Cell  , DOI: ( /S (03)00193-X)

7. Figure 6 Skp2 Promotes c-Myc-Induced Transcription
(A) Myc and Skp2 cooperatively activate the α-prothymosin (α-proT) promoter. HeLa cells were transiently cotransfected with the Myc-responsive α-proT promoter/luciferase (Luc) reporter gene, CMV-β-gal, and the indicated expression vectors. Promoter activity was measured by luciferase assays and normalized to β-gal activity (upper panel). The mut E promoter mutant lacks the Myc-responsive E-box. Exogenous and endogenous Myc levels were analyzed by immunoblot (lower panels).
(B) The transcriptional activities of Myc and Skp2 are interdependent. Myc−/− Rat1 and Skp2−/− MEF cells were transfected and analyzed as in (A).
(C) Inhibition of Myc and Skp2 expression reduces α-proT promoter activity. HeLa cells treated with the indicated siRNA duplexes were transfected with the α-proT promoter/reporter as described in the Experimental Procedures and analyzed as in (A) (left panel). Protein levels after siRNA treatment were determined by immunoblotting as indicated (right panels).
(D) Cell cycle arrest does not inhibit α-proT activation. HeLa cells were transfected with an α-proT promoter/luciferase (Luc) reporter gene, treated with indicated cell cycle blockers for 24 hr, and analyzed for luciferase activity as in (A).
(E) Activation of endogenous target gene expression by Myc and Skp2. p27−/− MEF MycER cells were transfected and OHT-treated as indicated. Cyclin D2 and ODC mRNA expression was quantitated using real-time RT-PCR. Results are presented as fold induction of mRNA expression.
Molecular Cell  , DOI: ( /S (03)00193-X)

8. Figure 7
Association of the c-Myc-Responsive Cyclin D2 Promoter with Skp2, Ubiquitylated Proteins, and Proteasome Components Is Myc Dependent
(A) Skp2 and Cul1 colocalize with c-Myc, ubiquitylated proteins, and proteasomal subunits on the cyclin D2 promoter. ChIP assays were performed using chromatin prepared from fixed HeLa cells using the indicated antisera or preimmune (p.i.) serum. PCR primers specific for the cyclin D2 promoter (upper panels) or the p21 coding region (lower panels) were used.
(B) Binding of Skp2, Sug1, and Ub to the cyclin D2 promoter is Myc dependent. ChIP assays were performed as in (A) using the indicated antibodies, and chromatin was prepared from fixed myc−/− and myc+/+ Rat1 cells. PCR primers specific for the promoters indicated were used.
(C) Expression of Skp2 in Rat1 cells. myc−/− and myc+/+ Rat1 cells were analyzed for expression of Skp2 by IP and Western. Skp2 overexpressed in Cos7 cells was included as control.
Molecular Cell  , DOI: ( /S (03)00193-X)