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Supporting Online Material for
SCFFbxl3 Controls the Oscillation of the Circadian Clock by Directing
the Degradation of Cryptochrome Proteins
Luca Busino, Florian Bassermann, Alessio Maiolica, Choogon Lee, Patrick M. Nolan,
Sofia I. H. Godinho, Giulio F. Draetta, Michele Pagano*
*To whom correspondence should be addressed. E-mail: [email protected]
Published 26 April 2007 on Science Express
DOI: 10.1126/science.1141194
This PDF file includes:
Materials and Methods
Figs. S1 to S10
References
Supporting Online Material
Materials and Methods
Cell culture, synchronization and drug treatment
Early-passage Cry1-/-;Cry2 -/- mouse embryonic fibroblasts (MEFs) (S1, S2), MEFs
from littermate wild type mice, NIH3T3 cells, HEK293T, and Hela S3 cells were
maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine
serum (FBS). To synchronize the circadian clock, NIH3T3 cells were grown to
confluence and then serum starved for 16 hours in the presence of 0.5% bovine
serum (BS). Serum starved cells were then shifted to medium containing 50% adult
horse serum and incubated for 90 minutes, after which they were washed twice with
PBS and cultured for the additional indicated hours in phenol-red free medium
containing 0.5% BS. Synchronization of MEFs was performed as for NIH3T3 cells,
except that the serum starvation step was omitted. To test the in vivo interaction
between FLAG-tagged F-box proteins and endogenous CRY proteins, the
proteasome inhibitor MG132 (Peptide Institute Inc.) (10 µM final concentration) was
added for 6 hours prior to harvesting the cells.
Purification and analysis of Fbxl3 interactors
Hela S3 cells were grown in suspension and retrovirally infected with a pBabe
FLAG-HA-Fbxl3 construct (see below). Approximately 5x109 cells were treated
with 10 µM MG132 for six hours. Cells were harvested and subsequently lysed in
lysis buffer (LB: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 50 mM
NaF, 0.5% NP40, plus protease and phosphatase inhibitors). Fbxl3 was
immunopurified with anti-FLAG agarose resin (Sigma) and washed 5 times with
LB (15 minutes each). After washing, proteins were eluted twice by competition
with FLAG peptide (Sigma). The eluate was then subject to a second
immunopurification with an anti-HA resin (12CA5 monoclonal antibody crosslinked to protein A Sepharose; Covance) prior to a competition with HA peptide
(Roche). The final eluate was separated by SDS-PAGE, and proteins were
visualized by silver staining. Bands were excised from the gel and digested
overnight in situ with Trypsin (Sigma). The acidified peptide solution was
desalted and concentrated as described (S3), injected via an Agilent 1100 Nano
HPLC onto a C18 column (Reprosil-Pur C18-AQ 3 µm), and packed into a spray
emitter (100 µm internal diameter, 8 µm opening, 70 mm length; New
Objectives). Peptides were eluted in a gradient from buffer A (5% acetonitrile
and 0.5% acetic acid) to buffer B (acetonitrile and 0.5% acetic acid) going from 0%
to 20% in 10 min at 300 nl min-1. Spectra were recorded on a QSTAR XL
quadrupole time-of-flight (TOF) tandem mass spectrometer
(AppliedBiosystems/MDS-Sciex).
Biochemical methods
Extract preparation, immunoprecipitation, and immunoblotting were previously
described (S4, S5). Mouse monoclonal antibodies were from Invitrogen (against
βTrcp1, Cul1, Skp1, Skp2, and Emi1), Sigma (anti-FLAG), Covance (anti-HA), BD
Transduction Laboratories (anti-c-Jun), Santa Cruz (against Actin, cyclin F, and
Skp1). Guinea pig polyclonal antibodies raised against Per1, Per2, Cry1, and Cry2
(S6) and the rabbit polyclonal anti-Cdk1 antibody (S7) were previously described.
2
The anti-Tim antibody was provided by P. Minoo (S8). The anti-Fbxl3 antibody was
produced in rabbit by immunization with a peptide corresponding to aa 228-236 (CIDDTPVDDP) of human Fbxl3. This region has high antigenic index and does not
show homology to other proteins in the database. A polyclonal antibody against
Fbxl10 was generated by immunizing rabbits with a peptide containing amino acids
800-1000 of human Fbxl10. Rabbit polyclonal antibody against Fbxl11 was raised
against a peptide containing amino acids 11-24 of human Fbxl11.
Plasmids and small hairpin RNAs
Per1, Per2, Cry1, Cry2, Clock, and Bmal1 cDNAs were provided by D.M. Virshup and
subcloned in pcDNA3.1. Tim cDNA was provided by J. Takahashi and subcloned
into pcDNA3.1. For retrovirus production, cDNAs encoding Fbxl3, βTrcp1, Skp2,
Fbxl2, Fbxl10, Fbxl11, Fbxl15, Emi1, Fbxo9, Fbx022, Cry1, and Cry2 were subcloned
into the retroviral vector pBabe and/or pallino (S9). Fbxl3 cDNA was amplified
using a 5’oligo encoding the FLAG-HA epitope tag sequence and subsequently
cloned in pBabe (S9). shRNAs targeting human and mouse Fbxl3 were constructed
in the Lenti-Lox3.7(pLL3.7) vector. The oligonucleotides used to construct the
shRNAs for human Fbxl3 were :
(#1) 5’ TGGACATTATTCTCCAAGTATTCAAGAGATACTTGGAGAATAATGTCCTTTTTTC 3’; and
(#2) 5’ TGGCCAACAATAGTGATACATTCAAGAGATGTATCACTATTGTTGGCCTTTTTTC 3’.
The oligonucleotides used to construct the shRNAs for mouse Fbxl3 were:
(#1) 5’ TGAATTGGAATCAGGTATTTTTCAAGAGAAAATACCTGATTCCAATTCTTTTTTC 3’;
(#2) 5’ TGGACAGCAGCAAAGAATCATTCAAGAGATGATTCTTTGCTGCTGTCCTTTTTTC 3’; and
(#3) 5’ TGCTTGTGAATTGCTCTTTATTCAAGAGATAAAGAGCAATTCACAAGCTTTTTTC 3’.
Transient transfections, retrovirus- and lentivirus-mediated DNA transfer
HEK293T cells were transfected using the calcium phosphate method as described
(S10). For retrovirus production, packaging GP-293 cells (Clontech) were
transfected with FuGENE-6 transfection reagent (Roche) according to the
manufacturer’s instructions. Forty-eight hours after transfection, the viruscontaining medium was collected and supplemented with 8 µg/ml polybrene
(Sigma). Cells were then infected by replacing the cell culture medium with the
viral supernatant for six hours. For lentivirus production, HEK293T cells were cotransfected with pLL3.7 and packaging vectors, and the virus-containing medium
was collected after 48 - 72 hours and supplemented with 8 µg/ml polybrene. Target
cells were incubated with the virus twice a day for three hours for two consecutive
days.
Recombinant proteins
cDNAs encoding the entire coding region of human Fbxl3 and Cry2 were inserted
into the baculoviral expression vector pFastBac HTb (Invitrogen). Bacterial vectors
for human Ubc3 (His-tagged) (S4), Ubc5 (His-tagged) (S4), and baculoviruses
expressing Skp2 (S7), βTrcp1, (S11), Skp1 (His-tagged), Cul1 (HA-tagged), and Roc1
(S7, S12) were previously described. All recombinant proteins were produced in 5B
insect cells as described (S13).
In vitro ubiquitylation assay
The ubiquitylation of Cry2 was performed in a volume of 10 µl containing 50 mM
Tris pH 7.6, 5 mM MgCl2, 0.6 mM DTT, 2 mM ATP, 1.5 ng/µl E1 (Boston Biochem),
3
10 ng/µl Ubc3, 10 ng/µl Ubc5, 2.5 µg/µl ubiquitin (Sigma), 1 µM ubiquitin
aldehyde, 2 µl of extracts from 5B insect cells expressing human Cry2, Skp1, Cul1,
Roc1, and one of the following human F-box proteins: Fbxl3, Skp2, or βTrcp1. In the
experiment shown in Fig. 2D, insect cell extracts were substituted with the indicated
immunoprecipitates. Where indicated, 2.5 µg/µl methylubiquitin (Boston Biochem)
was added instead of ubiquitin. The reactions were incubated at 30°C for the
indicated times and analyzed by SDS-PAGE and immunoblotting with an antibody
against Cry2.
mRNA Analysis
RNA was extracted using the RNeasy Kit (Qiagen). cDNA synthesis was performed
using Superscript III (Invitrogen). Quantitative PCR analysis was performed
according to standard procedures. Primer sequences were:
Per1, 5’ ACCTCAGCCAGCATCACCC 3’ and 5’ GAAGAGTCGATGCTGCCAAAG 3’
Per2, 5’ GTCCACCTCCCTGCAGACAAG 3’ and 5’ GCCTTCACCTGCTTCACGC 3’
Cry1, 5’ AAGAGGATGCCCAGAGTGTCG 3’ and 5’ CCTCCCACACGCTTTCGTATC 3’
18S rRNA, 5’ CGCCGCTAGAGGTGAAATTC 3’ and 5’ CTTTCGCTCTGGTCCGTCTT 3’
Chromatin immunoprecipitations
NIH3T3 cells were washed twice with PBS and cross-linked with 1% formaldehyde
at room temperature for 10 min. Cells then were rinsed with ice-cold PBS twice and
sonicated in lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1, and
protease inhibitor cocktail) three times for 10 seconds each, followed by
centrifugation for 10 minutes. Supernatants were collected and diluted in buffer (1%
Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl, pH 8.1) followed by
immunoclearing with 2 µg sheared salmon sperm DNA, 20 µl preimmune serum,
and protein G-sepharose for 2 hours at 4°C. Immunoprecipitations were performed
by incubating the supernatants overnight at 4°C with an anti-FLAG monoclonal
antibody. After immunoprecipitation, protein G-Sepharose, and 2 µg of salmon
sperm DNA were added, and the incubation was continued for 1 hour. Precipitates
were washed sequentially for 10 minutes each in buffer I (0.1% SDS, 1% Triton X100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl), buffer II (0.1% SDS, 1%
Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl), and buffer III
(0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1).
Precipitates were then washed three times with TE buffer and extracted three times
with 1% SDS, 0.1 M NaHCO3. Eluates were pooled and heated at 65°C for at least 6
hours to reverse the formaldehyde cross-linking. DNA fragments were purified with
a QIAquick Spin Kit (Qiagen). Quantitative PCR analysis was performed according
to standard procedure. Primers sequences were as reported in (S14):
Per1, 5'-CAGATGCCAGGAAGAGATCCTTAGCCAACC-3' and
5'-GACTAACCCTAGGATTGCAGCAGGGATCC-3';
Per2, 5'-AGCAGCATCTTCATTGAGGAACCCGGG-3' and
5'-CTCCGCTGTCACATAGTGGAAAACGTGAC-3';
Cry1, 5'-CTGCAACCAGCTCGGGCCGTC-3' and
5'-GATGAATGGGAGGCTGCCGAGGC-3'
rDNA, 5'-GGAGTGCGATGGTGTGATCT-3' and
5'-TAAAGATTAGCTGGGCGTGG-3'
4
Supplementary Figures
B
HA beads
176
97
176
97
66
56
47
66
56
47
36
36
31
31
*
FLAG-HA-Fbxl3
HA FT
HA eluate
HA beads
EV
FLAG eluate
FLAG-HA-Fbxl3
FLAG eluate
HA FT
HA eluate
HA FT
HA eluate
HA beads
FLAG eluate
EV
Immunoblot (anti-FLAG)
HA FT
HA eluate
HA beads
Silver Staining
FLAG eluate
A
*
*
*
*
Fbxl3
*
Fig. S1. Purification of the Fbxl3 complex. (A) FLAG-HA-tagged Fbxl3 was
retrovirally expressed in HeLa S3 cells and subjected to immunopurification using
anti-FLAG agarose and anti-HA resin. The FLAG peptide eluate (FLAG eluate),
the flow-through from the second column (HA FT), the HA peptide eluate (HA
eluate), and the SDS eluate of the material left on the HA resin (HA beads) were
separated by SDS-PAGE and visualized by silver staining. Bands were excised
from the lane containing the HA eluate and analyzed by mass spectrometry. The
mass spectrometry analysis of proteins co-purified with Fbxl3 revealed the
presence of two peptides (FLLQCLEDLDANLR and LTDLYKK) corresponding to
Cry1 and three peptides (GQPADVFPR, EFFYTAATNNPR, MKQIYQQLSR)
corresponding to both Cry1 and Cry2. As a control, a purification was performed
from HeLa S3 cells infected with an empty vector (EV). (B) 3% of the material
shown in (A) was analyzed by immunoblotting with an anti-FLAG antibody. The
asterisks indicate the immunoglobulin chains.
5
HA-Cry2
0
90
180
HA-Cry2 +
FLAG-Fbxl3
HA-Cry2 +
FLAG-Skp2
HA-Cry2 +
FLAG-Fbxl3 +
MG132
0
0
0
90 180
90 180
90
180
CHX (min)
Cry2 (anti-HA)
FBPs (anti-FLAG)
Actin
Fig. S2. Expression of Fbxl3 accelerates the degradation of Cry2. HEK293T cells
were transfected with vectors encoding HA-tagged Cry2 alone or HA-tagged
Cry2 in combination with either FLAG-tagged Fbxl3 or FLAG-tagged Skp2.
Three hours before lysis, cycloheximide (CHX) was added to the cell culture in
the presence or absence of MG132, as indicated. Extracts were then subjected to
immunoblotting with antibodies against HA (upper panels) to detect Cry2,
against FLAG (middle panels) to detect F-box proteins (FBPs), or against Actin
(bottom panels) to show normalization in loading.
6
B
Fblx3 shRNA 2
Fbxl3 shRNA 1
LacZ shRNA
UI
A
0 30 60 120 180 240 0 30 60 120 180 240 CHX(min)
Fbxl3 shRNA 1
Fbxl3 shRNA 2
Actin
2
3
Actin
LacZ shRNA
Fbxl3
1
Cry2 (anti-HA)
Fbxl3 shRNA 2
(short exp.)
4
Fig. S3. Downregulation of Fbxl3 stabilizes Cry2 in HEK293T cells. (A)
Retrovirally infected HEK293T cells expressing HA-tagged Cry2 were infected
with lentiviral constructs that direct the synthesis of shRNAs targeting LacZ (lane
2) or Fbxl3 (lanes 3 and 4, each expressing a different shRNA to human Fbxl3).
Lane 1 shows a cell extract from un-infected cells (UI). Twenty-four hours postinfection, cells were trypsinized, replated, and cultured for three additional days.
Finally, cells were collected, and protein extracts were probed with antibodies to
the indicated proteins. (B) The experiment was performed as in (A), except that
cycloheximide (CHX) was added for the indicated times. Notably, since the
transcription of exogenous Cry2 was driven by a heterologous promoter, the
increase in the level of Cry2 protein observed at time 0 using the shRNA 2 was
independent of the transcriptional regulation by Clock:Bmal1.
7
Fbxl3 shRNA 3
LacZ shRNA
Fbxl3 shRNA 1
Fbxl3 shRNA 2
A
Fbxl3
Actin
1
B
2
3
4
LacZ shRNA
+DMSO
Fbxl3 shRNA
+MG132
+DMSO
+MG132
Cry1 (anti-HA)
Actin
C
LacZ shRNA
+DMSO
Fbxl3 shRNA
+MG132
+DMSO
+MG132
Cry2 (anti-HA)
Actin
Fig. S4. Downregulation of Fbxl3 stabilizes CRY proteins in NIH3T3 cells. (A)
Retrovirally infected NIH3T3 cells expressing HA-tagged Cry1 were infected with
lentiviral constructs that direct the synthesis of shRNAs targeting LacZ (lane 1) or
Fbxl3 (lanes 2 - 4, each expressing a different shRNA to murine Fbxl3). Twentyfour hours post-infection, cells were trypsinized, replated, and cultured for three
additional days. Finally, cells were collected, and protein extracts were probed
with antibodies to the indicated proteins. (B) The experiment was performed as
in (A), except that cycloheximide (CHX) was added to the cell culture in the
presence or absence of MG132, as indicated. The shRNA construct 3, shown in
(A), was used. (C) The experiment was performed as in (B), except retrovirally
infected NIH3T3 cells expressing HA-tagged Cry2 were used.
8
Cry2
SCFFbxl3
Ubiquitin
Methylubiquitin
.
.
.
.
in in in. in in. in
m
m
m
m
45 45 5m 45 5m 45
+ + + + + +
+ + + + +
+ +
+ +
Cry2
(Ub)n
Cry2
Fig. S5. The high molecular weight forms of Cry2 are polyubiquitylated
species of the protein. In vitro ubiquitin ligation assays of recombinant Cry2
protein were conducted in the presence of recombinant SCFFbxl3. Samples were
incubated at 30°C for the indicated time, and reactions were then stopped by the
addition of Laemmli sample buffer. Where indicated, methylated ubiquitin was
added to the reaction mix. Finally, samples were analyzed by SDS-PAGE and
immunoblotting analysis using an antibody against Cry2. The bracket on the left
side of the panel marks a ladder of bands corresponding to polyubiquitylated
Cry2. When methylated ubiquitin (which is chemically modified to block all of
its free amino groups) was added to the in vitro reaction, the abundance of the
slowest migrating bands decreased. This effect is due to the capping of
polyubiquitin chains by methylated ubiquitin that competes with unmodified
ubiquitin and terminates the polyubiquitin chains. This experiment formally
demonstrates that the high molecular weight forms of Cry2 are
polyubiquitylated species of the protein.
9
Fbxl3 shRNA
Asynch.
50% HS
6
12
18
24
30
36
42
48
54
Asynch.
50% HS
6
12
18
24
30
36
42
48
54
A
LacZ shRNA
hours post-serum shock
hours post-serum shock
Cry1 (long exp.)
Cry1 (short exp.)
Per1
Per2
Fbxl3
c-Jun
Tim1
Actin
B
hours post-serum shock
250
200
150
100
50
0
36
42
48
54
24
30
Cry1
Per1
Per2
y
50 nch
% .
H
S
6
12
18
fold increase
Fbxl3 shRNA
As
250
200
150
100
50
0
As
y
50 nch
% .
H
S
6
12
18
24
30
36
42
48
54
fold increase
LacZ shRNA
hours post-serum shock
Fig. S6. Fbxl3 promotes the oscillations of Cry1 and the accumulation of PER
proteins. (A) NIH3T3 fibroblasts were infected with lentiviral constructs that
direct the synthesis of shRNAs targeting LacZ or Fbxl3, as indicated. In this
experiment the shRNA construct 3 to mouse Fbxl3 (see fig. S4A) was used. Cells
were asynchronously grown to confluence in a medium containing 10% fetal
bovine serum (Asynch.). Asynchronous cells were serum starved for 16 hours and
then shifted to a medium containing 50% serum for 90 minutes (50% HS). After
this period, the serum-rich medium was replaced with 0.5% serum-containing
medium and cells were collected at the indicated hours post-serum shock. Cell
extracts were analyzed by immunoblotting with antibodies to the indicated
proteins. The experiment with this particular shRNA construct to mouse Fbxl3
was repeated two times with similar results. (B) The graphs illustrate the
quantification by densitometry of the results shown in (A). The value given for
the amount of Cry1, Per1, and Per2 present in asynchronous cells treated with
shRNAs targeting LacZ was set as 100%.
10
A
y
As
LacZ shRNA
Fbxl3 shRNA nc
+
h.
%
50
SD
+
+
HS Hours post-serum shock
8
4
20
12
+
+
+
+
+
+
+
+
+
+
+
Cry1
Per2
Fbxl3
Actin
B
shRNA LacZ
shRNA Fbxl3
120
Per2 protein
(fold increase)
80
60
40
20
100
50
20
12
8
4
H
S
.
D
.
ch
S.
50
%
Hours post-serum shock
yn
20
12
8
4
H
S
D
.
50
%
S.
.
ch
yn
150
0
0
As
200
As
Cry1 protein
(fold increase)
100
Hours post-serum shock
Fig. S7. Fbxl3 promotes the accumulation of Per2 and controls oscillations of
the clock. (A) NIH3T3 fibroblasts were infected with lentiviral constructs that
direct the synthesis of shRNAs targeting LacZ or Fbxl3, as indicated. In this
experiment the shRNA construct 2 to mouse Fbxl3 (see fig. S4A) was used. Cells
were asynchronously grown to confluence in a medium containing 10% fetal
bovine serum (Asynch.). Asynchronous cells were serum deprived for 16 hours
(SD) and then shifted to a medium containing 50% serum for 90 minutes (50%
HS). After this period, the serum-rich medium was replaced with 0.5% serumcontaining medium and cells were collected at the indicated hours post-serum
shock. Cell extracts were analyzed by immunoblotting with antibodies to the
indicated proteins. The experiment with this particular shRNA construct to
mouse Fbxl3 was repeated two times with similar results. (B) The graphs
illustrate the quantification by densitometry of the results shown in (A). The
value given for the amount of Cry1 and Per2 present in asynchronous cells
treated with shRNAs targeting LacZ was set as 100%.
11
LacZ shRNA
Fbxl3 shRNA
3
2
1
0
SD
50% HS
3
6
9
12
15
18
21
24
27
30
33
36
39
42
45
48
Per1 mRNA
(fold increase)
4
hours post-serum shock
3
2
1
0
SD
50% HS
3
6
9
12
15
18
21
24
27
30
33
36
39
42
45
48
Per2 mRNA
(fold increse)
4
hours post-serum shock
3
2
1
0
SD
50% HS
3
6
9
12
15
18
21
24
27
30
33
36
39
42
45
48
Cry1 mRNA
(fold increase)
4
hours post-serum shock
Fig. S8. Fbxl3 promotes the accumulation of Clock:Bmal1-regulated mRNAs.
The experiment was performed as in Fig. S7, except that whole-cell RNA was
prepared to determine the levels of the indicated mRNAs using quantitative RTPCR. The value given for the amount of mRNA present in asynchronous cells
treated with shRNAs targeting LacZ was set as 1. Error bars represent + SD
(n=3).
12
*
Fold Increase
5
*
*
UI
LacZ shRNA
Fbxl3 shRNA
4
3
2
1
0
Per2
promoter
Per1
promoter
Cry1
promoter
rDNA
promoter
Fig. S9. Stabilized Cry1 binds the promoters of Clock:Bmal1-regulated genes.
Retrovirally infected NIH3T3 cells expressing FLAG-tagged Cry1 or un-infected
NIH3T3 cells (UI) were infected with lentiviral constructs carrying shRNAs
against LacZ or Fbxl3 and subsequently used for chromatin
immunoprecipitations using an anti-FLAG antibody. DNA precipitated with the
anti-FLAG antibody was amplified by real-time PCR using primers flanking the
indicated gene promoters. The unrelated rDNA promoter was used as negative
control. The value given for the amount of PCR product present in un-infected
NIH3T3 cells was set as 1. Error bars represent + SD (n=3). Asterisks indicate a p
value <0.01 measured using a t-test.
13
LacZ shRNA
Fbxl3 shRNA
HA-Cry2
HA-Cry2
HA-Cry2
+ Fbxl3
0 1 3 5
0 1 3 5
0 1 3 5
HA-Cry2
+Fbxl3
(C358S)
0 1 3 5
CHX (hours)
Cry2 (anti-HA)
Cry2 (anti-HA) (short exp.)
Fbxl3
Actin
Fig. S10. Fbxl3(C358S) does not induce degradation of Cry2. NIH3T3 cells were
infected with retroviral vectors encoding HA-tagged Cry2 alone or HA-tagged
Cry2 in combination with either FLAG-tagged human Fbxl3 or FLAG-tagged
human Fbxl3(C358S), as indicated. Cells were then infected with lentiviral
constructs that direct the synthesis of shRNAs targeting LacZ or mouse Fbxl3
(which does not target retrovirally expressed human Fbxl3). Twenty-four hours
post-infection, cycloheximide (CHX) was added to the cell culture for the
indicated times. Finally, cells were collected, and protein extracts were probed
with antibodies to the indicated proteins. Human Fbxl3 (which is insensitive to
mouse shRNA) reverses the stabilization of Cry1 induced by the shRNA
targeting mouse Fbxl3. In contrast, expression of human Fbxl3(C357A) (which is
also insensitive to mouse shRNA) is unable to induce the destabilization of Cry1,
proving that this mutant is less active than wild-type Fbxl3.
14
References
S1.
S2.
S3.
S4.
S5.
S6.
S7.
S8.
S9.
S10.
S11.
S12.
S13.
S14.
G. T. van der Horst et al., Nature 398, 627-30 (1999).
L. P. Shearman et al., Science 288, 1013-9 (2000).
J. Rappsilber, Y. Ishihama, M. Mann, Anal Chem 75, 663-70 (2003).
A. Peschiaroli et al., Mol Cell 23, 319-29 (2006).
N. V. Dorrello et al., Science 314, 467-71 (2006).
C. Lee, J. P. Etchegaray, F. R. Cagampang, A. S. Loudon, S. M. Reppert,
Cell 107, 855-67 (2001).
A. C. Carrano, E. Eytan, A. Hershko, M. Pagano, Nat Cell Biol 1, 193-199
(1999).
J. Xiao, C. Li, N. L. Zhu, Z. Borok, P. Minoo, Dev Dyn 228, 82-94 (2003).
A. C. Carrano, M. Pagano, J Cell Biol 153, 1381-1389 (2001).
T. Bashir, N. V. Dorrello, V. Amador, D. Guardavaccaro, M. Pagano,
Nature 428, 190-3 (2004).
L. Busino et al., Nature 426, 87-91 (2003).
F. Bassermann et al., Cell 122, 45-57 (2005).
A. Montagnoli et al., Genes & Dev 13, 1181-1189 (1999).
J. P. Etchegaray et al., Nature 421, 177-82 (2001).
15