SUPPLEMENTARY FIGURE LEGENDS
FIGURE S1: E2F family proteins are NEDDylated in cells.
A–F. HEK293T cells were transfected with GFP along with a plasmid encoding either
His6-tagged ubiquitin (His-Ub), NEDD8 (His-NEDD8), or the corresponding empty vector,
along with a plasmid for each protein as indicated. Total cell lysates (bottom panels) as well
as His6-tagged proteins purified there from (top panels) were subjected to immunoblot
analysis with antibodies to the indicated proteins (bottom panels) or to the test protein (top
panels). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and GFP were examined as
loading and transfection controls, respectively. 3×FLAG- pRB and HA-HAUSP was detected
with antibodies to FLAG and HA, respectively. Asterisks represent nonspecifically
precipitated proteins.
G. HEK293T cells were transfected with a plasmid encoding 3×FLAG-tagged forms of
ubiquitin or NEDD8, after which cell lysates were prepared and subjected to immunoblot
analysis with antibodies to FLAG.
H. HEK293T cells were transfected with a plasmid encoding His-NEDD8, or the
corresponding empty vector. 18 h after transfection, cells were treated with 1 μM MLN4924
and incubated for 6 h. Cells were analyzed as described in A-F. MLN4924 was purchased
from Active Biochemicals Co.
FIGURE S2: E2F family proteins are deNEDDylated by SENP8.
A–D. HEK293T cells were transfected with plasmids encoding GFP and His6-NEDD8, along
with a plasmid encoding each protein as indicated, and either a plasmid encoding HA-tagged
SENP8 or the corresponding empty plasmid. Cell lysates and His6-tagged proteins purified
from there were then subjected to immunoblot analysis with antibodies to the indicated
proteins. Asterisks indicate nonspecifically precipitated proteins.
E. HEK293T cells were transfected with a plasmid encoding 3×FLAG-tagged forms of
ubiquitin or NEDD8 and with a plasmid for HA-SENP8 as indicated. Cell lysates were
subjected to immunoblot analysis with antibodies to FLAG..
FIGURE S3: NEDDylation of E2F1 is attenuated in response to DNA damage in a
SENP8-dependent manner
A. H1299 cells were transfected first with SENP8 or control siRNAs (10 nM) for 24 h and
then with plasmids for His6-NEDD8, for GFP, and for E2F1 for 24 h. Cell lysates as well as
His6-tagged proteins purified from there were subjected to immunoblot analysis with
antibodies to E2F1. The asterisk indicates a nonspecifically precipitated protein.
B. U2OS cells were transfected first with SENP8 or control siRNAs (5 nM) for 24 h and then
with plasmids for His6-NEDD8, for GFP, and for E2F1 for 24 h. The cells were then
incubated in the absence or presence of 2 μM doxorubicin for 12 h, after which cell lysates as
well as His6-tagged proteins purified from there were subjected to immunoblot analysis with
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antibodies to E2F1. The asterisk indicates a nonspecifically precipitated protein.
FIGURE S4: SENP8 is dispensable for E2F1’s ability to promote cell cycle.
SAOS-2 cells stably expressing ER-E2F1 were transfected with SENP8 or control siRNAs (5
nM) for 48 h and then incubated in the absence or presence of 500 nM 4-OHT for 12 h. Cells
were then collected and analyzed for cell cycle profile by flow cytometry.
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SUPPLEMENTARY MATERIALS
Plasmids
pT7-7-His6-Ubc12 and pT7-7-HA-His6-NEDD8GG were kindly provided by K. Iwai;
pT7-T-UbcH5b and pGEX6P1-Ub by K. Tanaka; pRc/CMV-HA-E2F1, -E2F2, -E2F3, and
-E2F4 as well as the E2F2-luciferase reporter construct by A. Zubiaga;
pcDNA3.1-3×FLAG-RB by M. Kitagawa; pCI-FLAG-PCAF by K. Ishiguro;
pcDNA3-HA-YY1 by M. Miyagishi; pcDNA3-HA-p300 by Y. Mori; and pCG-N-HA-Jab1 by
J. Kato. The pCAGEN-His6 vector was constructed by ligating the hybridized
oligonucleotides 5′-AATTATGGGGGGTTCTCATCATCATCATCATCATGAATTCC-3′ and
5′-TCGAGGAATTCATGATGATGATGATGATGAGAACCCCCCAT-3′ into the EcoRI-XhoI
sites of pCAGEN. NEDD8 cDNA was amplified from HeLa cell cDNA and cloned in
pCAGEN-His6 or in frame with the 3×FLAG tag in pCAGEN. SENP8 cDNA was amplified
from HeLa cell cDNA and cloned in frame with the HA or 3×FLAG tags in pCAGEN.
HAUSP cDNA was amplified from HeLa cell cDNA and cloned in frame with the HA tag in
pCAGEN. DP1 cDNA was amplified from HeLa cell cDNA and cloned in frame with the
FLAG tag in pcDNA3.1 (Invitrogen). A DNA fragment encoding chimpanzee ubiquitin was
isolated from pGEX6P1-Ub and subcloned into the EcoRI-XhoI sites of pCAGEN-His6 or in
frame with the 3×FLAG tag in pCAGEN. MCPH1 cDNA was amplified from HeLa cell
cDNA and cloned in frame with the 3×FLAG tag in pCAGEN. Slc7a1 cDNA was amplified
from mouse embryonic fibroblast cDNA and cloned in frame with the HA tag in pcDNA 3.1.
A DNA fragment encoding human E2F1 was isolated from pRc/CMV-HA-E2F1 and
subcloned into the BamHI-NotI sites of pcDNA3 (Invitrogen) or of pCMV/SV2-GAL4DBD
(kindly provided by H. Sasaki). Mutagenesis was performed by PCR according to standard
protocols. A DNA fragment corresponding to amino acids 1 to 368 of E2F1 was amplified by
PCR from pcDNA3-E2F1 and subcloned together with DNA encoding the TAD of VP16 into
the BamHI-EcoRI sites of pCS2. The p73 gene promoter (nucleotides –4091 to +438 relative
to the transcription start site) was amplified by PCR from the human genome BAC clone
RP5-1092A11 (BACPAC Resources) and cloned into the MluI-NheI sites of pGL3-basic
(Promega).
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Antibodies
Antibody
FLAG (M2)
M2 Affinity Gels
E2F1 (C-20)
E2F2 (C-20)
E2F3 (C-18)
E2F4 (A-20)
DP1 (K-20)
GFP (598)
HAUSP
Cleaved PARP (#9541)
SENP8
p53 (Ab-8)
HA (3F10)
GAPDH (6C5)
pRB (G3-245)
Caspase-3 (19)
p73 (5B429)
Cyclin E (HE12)
NEDD8 (Y297)
Company
Sigma
Sigma
Santa Cruz Biotechnology
Santa Cruz Biotechnology
Santa Cruz Biotechnology
Santa Cruz Biotechnology
Santa Cruz Biotechnology
MBL
Bethyl
Cell Signaling
Enzo Life Science
Calbiochem
Roche
Chemicon
BD Pharmingen
BD Pharmingen
Imgenex
Santa Cruz Biotechnology
Epitomics
Primer sequences for real-time PCR
Gene
Forward
PPIA
TTGCTGACTGTGGACAACTC
p73
CAGACAGCACCTACTTCG
NOXA
CGAAGATTACCGCTGGC
SENP8
TGATTCCCATAGCAGGAGC
CCNE1
AGCCTTGGGACAATAATGC
CDC25A
CCTACTGATGGCAAGCG
E2F2
CCTGACCTCAAGTGATCC
AXIN2
TGGACCAAGTCCTTACACTC
Reverse
ACAAAGATTCTAGGATACTGCGA
GACGTCCATGCTGGAATC
GCAACAACAACAATGCACT
GGCCAGTTTGTCTCCTT
GCACGTTGAGTTTGGGTAA
GGCGATCTCTCTCTCTCACATA
GTCTACCTGGTCCCTAAAGAAA
GCAAACCAGAAGTCTAAGGTATC
SUPPLEMENTARY METHOD
Quantification of Proteins
The bands resulting from immunoblotting were quantified with the use of ImageQuant LAS
and ImageQuant TL software (GE Healthcare) according to the manufacturer’s protocol.
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