complexes from cultured cells. Reactions were stopped with
sample loading buffer. Samples were separated by SDS-PAGE
P uptake was detected by autoradiography.
Reconstitution of cyclin D1 polyubiquitination
were prepared from transfected
HEK293 cells. Equal amounts of SCFL
were mixed with 1 mg GST-full-length Cyc D1 WT protein in the
presence of 30 ng recombinant active ERK2 (Upstate) and
0.5 mM ATP for 30 min on ice to allow binding. To the mixture
was added 50 ng E1, 100 ng E2 (UbcH5c), 2 mg ubiquitin, and
1 mg ubiquitin aldehyde (all from BostonBiochem). Reactions were
performed with a buffer containing 50 mM Tris-HCl (pH 8.0),
1 mM DTT, 5 mM MgCl
, 0.5 mM EDTA, 1.5 mM ATP in the
presence of 10% glycerol at 30uC for 1 hour and terminated by
boiling for 5 min with SDS sample loading buffer. Samples were
separated by SDS-PAGE and immunoblotted with cyclin D1
antibody (A-12, Santa Cruz).
Figure S1 (A, B) Expression profile of cyclin D1 during cell cycle
progression after release from quiescence. WI-38 cells (A) and
T98G cells (B). (C) Western blot analysis using nuclear (N) and
cytoplasmic (C) fraction (Fr.) proteins extracted from cell lysates
collected in Figure 1F, lane 3. The membrane was stained with
histone H1, MEK1, and cyclin D1. (D) Immunoprecipitation-
immunoblot analysis. HCT 116 cells were transfected with
ubiquitin cDNA and synchronized to S phase through sequential
manipulation of serum starvation and stimulation. Cells were
treated with Leptomycin B (LMB) for 3 hours to inhibit nuclear-
to-cytoplasmic localization of cyclin D1 and treated with MG132
for 1 hour before harvesting. Nuclear protein (N) was fractionated
and immunoprecipitated with a cyclin D1 antibody and
immunoblotted with a HA antibody (upper panel) or a cyclin
D1 antibody (lower panel). Asterisk: background non-specific
bands. (E) In vitro ubiquitination assay. In vitro translated F-box
proteins with recombinant GST-b-catenin (Upstate), HeLa cell
extracts Fraction II with ATP, Ubiquitin and GSK3b, and in vitro-
translated SKP1, RBX1 and CUL1 were incubated at 30u C for
2 hours. Samples were separated by SDS-PAGE and immuno-
blotted with a b-catenin antibody. (F) Immunoblot analysis. HCT
116 cells were infected with a retrovirus expressing FBXW7/
CDC4 or a control retrovirus expressing GFP. Cells were
harvested 48 hrs and Western blot analysis was performed with
antibodies to cyclin D1, cyclin E, Flag (FBXW7), GFP and CDK4.
(G) Summary of RT-PCR following depletion of CUL1, CUL7 or
FBXW8 expression for 48 hrs through siRNA or mismatch (MM)
oligonucleotides in HCT 116 cells (see Fig. 4E). Non-targeting
siRNA was provided as control. Relative gene expression is shown.
(H, I) Expression profile of cyclin D3 protein during cell cycle
progression after release from quiescence. WI-38 cells (H) and
HCT 116 cells (I).
Found at: doi:10.1371/journal.pone.0000128.s001 (2.44 MB TIF)
The authors gratefully acknowledge Drs. Allan Balmain, Mike Fried, Eiji
Hara, Demetris Iacovides, Anthony Karnezis, Haruhiko Koseki, Valerie
Natale, David Stokoe and Thea Tlsty for their valuable suggestions and
Conceived and designed the experiments: OT HO. Performed the
experiments: OT HO SL JP. Analyzed the data: FM OT HO SL JP.
Contributed reagents/materials/analysis tools: FM DA OT HO. Wrote
the paper: OT.
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PLoS ONE | www.plosone.org 11 December 2006 | Issue 1 | e128