p120 Acts as a Specific Coactivator for 9-cis-Retinoic Acid Receptor (RXR) on Peroxisome Proliferator-Activated Receptor-
/RXR Heterodimers
Tsuyoshi Monden,
Mikiko Kishi,
Takeshi Hosoya,
Teturou Satoh,
Fredric E. Wondisford,
Anthony N. Hollenberg,
Masanobu Yamada and
Masatomo Mori
First Department of Internal Medicine (T.M., M.K., T.H., T.S.,
M.Y., M.M.) Gunma University School of Medicine Maebashi
371 Japan
Thyroid Unit (F.E.W., A.N.H.) Department of
Medicine Beth Israel Deaconess Medical Center and Harvard Medical
School Boston, Massachusetts 02215
 |
ABSTRACT
|
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p120 was originally isolated as a novel nuclear
coactivator for thyroid hormone receptor. In this study, we
characterized its interaction and transactivation of peroxisome
proliferator-activated receptor-
(PPAR
) and
9-cis-retinoic acid receptor (RXR) heterodimers. Transient
transfection study revealed that p120 enhanced the transcriptional
activation of PPAR
/RXR induced by PPAR
- or RXR-specific ligands.
In the glutathione-S-transferase pull-down assay, while
steroid receptor coactivator-1 showed apparent interactions with both
RXR and PPAR
, p120 bound only to RXR in a 9-cis-retinoic
acid (RA)-dependent manner and also did not bind to PPAR
even
in the presence of thiazolidinediones. The yeast two-hybrid
analysis showed no interaction of p120 with PPAR
under any
conditions, and electophoretic mobility shift assay showed apparent
DNA-PPAR
/RXR/p120 complex formation only in the presence of
9-cis-RA. Furthermore, the yeast three-hybrid assay clearly
revealed a significant interaction between p120 and PPAR
via RXR of
PPAR
/RXR heterodimer only in the presence of 9-cis-RA.
These findings indicate that p120 acts as a specific coactivator for
the RXR of PPAR
/RXR heterodimer in a 9-cis-RA-dependent
manner.
 |
INTRODUCTION
|
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We have recently cloned p120 using a yeast two-hybrid system and
the ligand-binding domain of the thyroid hormone receptor (TR) ß1 as
bait (1). The amino acid sequence deduced from its cDNA sequence
revealed that p120 consists of 920 amino acids, and part of the
sequence was identical to that of a previously identified protein,
skeletal muscle abundant protein (SMAP) (2). However, Northern analysis
demonstrated that p120 was expressed not only in the skeletal muscle
but also in various organs. p120 was shown to interact
specifically with the AF-2 domain of TR, and the interacting domains of
p120 were localized in the region between amino acids (AA) 186297.
Transient transfection study revealed that p120 enhances the
transcriptional activation by TR in a thyroid hormone-dependent manner.
Therefore, in this context, p120 is similar to the steroid receptor
coactivator-1 (SRC-1). However, subsequent examination showed p120
functions that differed from those of SRC-1: while SRC-1 is a
coactivator for most nuclear receptors, including glucocorticoid
receptor (GR), progesterone receptor (PR), TR, estrogen receptor (ER),
androgen receptor (AR), vitamin D receptor (VitDR), retinoic acid
receptor (RAR) and 9-cis-retinoic acid receptor (RXR) (3),
p120 acts as a coactivator for AR but not for ER or RAR.
Peroxisome proliferator-activated receptor (PPAR) also is
a member of the nuclear receptor superfamily (4). To date,
three isoforms of PPARs have been identified (PPAR
, -
, and -
),
and they exhibit different tissue distribution patterns (5).
Furthermore, PPAR
has two isoforms, PPAR
1 and
2, which share a
high degree of homology except in the N-terminal region (6). These
isoforms also showed different tissue distributions from PPAR
1,
which is expressed ubiquitously, and PPAR
2, which is
expressed predominantly in adipose tissue, where it has been shown
to play an important role in adipocyte differentiation (7, 8). The
actions of this receptor are known to be regulated by
thiazolidinediones, a class of antidiabetic reagent (9), and the
fatty acid derivative 15-deoxy-PGJ2 (10, 11), which bind to PPAR
and
promote adipogenesis. Recently, oxidized low-density lipoprotein (12, 13) was reported to be an endogenous ligand of PPAR
and was
suggested to be related to atherosclerosis (14).
When these ligands activate the PPAR
response gene, PPAR
heterodimerizes with RXR, a common heterodimer partner with TRs, RAR,
and VitDR (15, 16), and this heterodimer binds to the PPAR-response
elements (PPREs) in target genes to activate transcription (17, 18).
The PPRE has been identified as a direct repeat motif of hexamer
half-sites, TGACCT, spaced by one nucleotide (DR1) (19, 20), and has
been found in various genes including the peroxisomal ß-oxidation
enzymes (21), lipoprotein lipase (19), acyl-CoA oxidase (22),
phospoenlpyruvate carboxykinase (23), aP2 (24), UCP-1 (25), and leptin
(26). Furthermore, it has been reported that this activation of gene
transcription by PPAR/RXR is dependent on the recruitment of
coactivators such as SRC-1 (27, 28), PPAR
coactivator-1 (PGC-1) (29)
and PPAR
-binding protein (PBP) (30). All of these coactivators
interact with PPAR
in either a ligand-dependent or independent
manner. In the present study, we examined whether p120 functions as a
coactivator for PPAR
and determined the mechanism by which p120
interacts with PPAR
/RXR. We demonstrated that p120 acts as a
coactivator for PPAR
/RXR and predominantly interacted with the RXR
on PPAR
/RXR heterodimer complex only in the presence of
9-cis-RA, suggesting that p120 mediates enhancement of
PPAR
/RXR transcriptional activation through a different mechanism
from other coac-tivators.
 |
RESULTS
|
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p120 Is Expressed in Human Fat Tissue
We initially examined the expression of p120 mRNA in human fat
tissue. While our previous study showed the ubiquitous expression of
the p120 gene in various tissues (1), the expression of p120 in fat
tissue was unknown. Therefore, we performed RT-PCR analysis to confirm
the expression of p120 in fat tissue. Lane 2 of Fig. 1
shows the expression of the p120 mRNA
in human fat tissue. However, SRC-1 mRNA was not detectable in the fat
tissue under the same conditions (Fig. 1
, lane 4). This result suggests
that p120 may play a role in adipocyte differentiation or lipid
metabolism along with PPAR
.

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Figure 1. Expression of p120 Gene in Fat Tissue
Lanes 1 and 2 were hybridized with 32P-labeled p120
cDNA, and lanes 3 and 4 were hybridized with 32P-labeled
SRC-1 cDNA as probes. The following samples were used at each lane as a
template: lane 1, p120 cDNA; lanes 2 and 4, cDNA from human fat tissue;
lane 3, SRC-1 cDNA. The primers were described in Materials and
Methods.
|
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p120 Acts as a Coactivator of PPAR
/RXR in CV-1 Cells
To examine whether p120 acts as a coactivator of PPAR
/RXR in
the presence of specific ligands, we performed transient transfection
assays using CV-1 cells transfected with PPAR
2 and RXR
expression
vector. As shown in the left panel of Fig. 2
, incubation with
9-cis-retinoic acid (9-cis RA) showed the minimum
increase (
2-fold) of the basal activity of the parental vector
containing the TK109 promoter, whereas troglitazone (TZ) did not cause
any changes. In this system, expression of p120 did not affect the
luciferase activity of the thymidine kinase (TK) promoter in either the
absence or presence of ligands, suggesting that p120 did not enhance
general transcription.
In contrast, 1 µM 9-cis-RA and 10
µM TZ caused 8- and 5-fold activation of the TK promoter
activity fused to the DR1 element (Fig. 2
, right panel),
respectively, and synergistic activation (19-fold) was observed by
adding both ligands simultaneously. Cotransfection of p120 was able to
augment each ligand-dependent activation of DR1 reporter expression
(28-fold, 9-cis-RA; 14-fold, TZ). The synergistic
effect of both ligands was further stimulated by expression of p120
(72-fold). Furthermore, expression of p120 alone enhanced the
RXR/PPAR
-mediated activation of DR1 reporter expression by 3.4-fold
(Fig. 2
). This result suggested the presence of endogenous
9-cis-RA-like activity in this culture system, and an
equally likely senario is that p120 is interacting with RXR in a
ligand-independent manner under these experimental conditions. These
observations demonstrated that p120 functions as a coactivator of
PPAR
/RXR heterodimer in the presence of specific ligands.
p120 Interacts with RXR
but Not with PPAR
To understand the mechanisms by which p120 activates PPAR
/RXR,
we studied the protein-protein interactions among these three molecules
using glutathione-S-transferase (GST) pull-down assay and
the yeast two-hybrid assay. We previously demonstrated that the first
297 amino acids of p120 and AA 186297 of p120 containing the LXXLL
motif are essential for the interaction with TR in a ligand-dependent
manner; therefore, we used these regions to study its interaction with
RXR
or PPAR
. As shown in Fig. 3
, the interacting domain of SRC-1, containing three LXXLL motifs, clearly
interacted with RXR
in a 9-cis-RA-dependent manner and
with PPAR
in either the presence or absence of ligand. In contrast,
AA 1297 of p120 interacted with RXR
only in the presence of
9-cis-RA, and AA 186297 of p120 showed stronger
interaction with RXR
. Furthermore, no interaction was detected
between p120 and PPAR
in either the presence or absence of TZ (Fig. 3
). Similar results were observed when AA 1920 (the whole protein)
for AA 1297 of p120 was used (Fig. 3
).
To confirm the above results, we next examined whether PPAR
interacted with p120 using the yeast two-hybrid assay. As shown in Fig. 4A
, although the ligand-binding domain of
PPAR
including the hinge region strongly interacted with RXR
in
either the presence or absence of TZ, PPAR
was not able to interact
with any derivatives of p120 that covered all coding regions of p120
even in the presence of TZ. In the same yeast system, p120 interacted
with RXR in a 9-cis-RA-dependent manner and also this data
suggested that AA 186297 of p120 are important for the interaction
with RXR as well as TR (Fig. 4B
).
Taken together, these observations suggested that the activation
PPAR
/RXR by p120 may be caused by the direct protein-protein
interaction of PPAR
-RXR but not by PPAR
-p120.
Detection of p120-RXR
-PPAR
Complex on the DR1 DNA Element
To confirm the results obtained in the above in vitro
assays, we next tested whether p120 could interact with PPAR
and
RXR-DNA complex using electrophoretic mobility shift assay (EMSA) with
the bacterially expressed AA 186297 of p120, in vitro
translated PPAR
2, RXR
, and labeled DR1 probe. Our previous study
showed that this domain of p120 bound to TR homodimers and TR/RXR
heterodimers on the DR4 element in the presence of thyroid hormone. As
shown in Fig. 5
, PPAR
did not form
either monomer or homodimers on the DR1 element even in the presence of
p120. On the other hand, RXR
formed homodimers on the DR1 element,
and addition of 9-cis-RA promoted this homodimerization.
Furthermore, addition of GST-p120 (AA 186297) supershifted this
complex only in the presence of 9-cis-RA, indicating the
formation of the p120/RXR/RXR complex in the presence of
9-cis-RA. As expected, PPAR
and RXR
heterodimer
complex strongly bound to the DR1 elements, and addition of p120
supershifted PPAR
/RXR
heterodimer complex only in the presence of
9 cis-RA or a combination of TZ and 9-cis-RA.
While the p120/RXR/RXR complex and the p120/RXR
/PPAR
complex were
not distinct because these complexes showed the same size on the gel,
addition of p120 made the RXR
/PPAR
heterodimer faint, and
the RXR/RXR homodimers still remained, strongly suggesting that these
supershifts were formed mainly by the p120/RXR
/PPAR
complex.
However, this supershift was not observed after addition of TZ alone,
indicating that p120 forms a ternary complex with PPAR
-RXR
heterodimer on PPREs, which is 9-cis-RA dependent.
Furthermore, the GST proteins alone did not bind to the DR1 probe.
The Yeast Three-Hybrid Assay Demonstrates that 9-cis-RA
Promotes the Interaction of p120 with the RXR on PPAR
/RXR
Heterodimers
To examine specific interactions of p120 with the RXR on
PPAR
/RXR heterodimers, we next performed yeast three-hybrid assay
between the GAL4 DNA-binding domain-PPAR
fusion and GAL4 activation
domain-p120 fusion in the presence or absence of RXR
expression
(Fig. 6
). For this system, we fused cDNAs
of RXR
, PPAR
, and p120 into the pAUR123, a
yeast-Escherichia coli shuttle vector (Takara, Berkeley,
CA), pGBT9 vector, and pGAD24 vector (CLONTECH Laboratories, Inc., Palo Alto, CA), respectively, all of which were driven by
the alcohol dehydrogenase 1 promoter (ADH1) (31). Under the control of
ADH1 promoter, the heterologous protein is expressed at a high level in
yeast cells during growth on glucose-rich medium (32). We then
transformed pGBT9-PPAR
with the pAUR123-RXR and/or pGAD-P120 into
HF7c yeast cells and plated them on plates lacking leucine, tryptophan,
and histidine. As shown in the left panel of Fig. 6A
, in the
absence of both p120 and RXR, addition of 9-cis-RA or TZ had
no effect on basal ß-galactosidase activity. Cotransfection of RXR
alone also showed no effect (second panel). On the other
hand, in the presence of p120 (AA 1297), TZ and 9-cis-RA
showed a slight increase in ß-galactosidase activity in the absence
of RXR, but these changes were not significant. In contrast, in the
presence of RXR and p120, while TZ alone did not cause significant
changes in ß-galactosidase activity, 9-cis-RA promoted
activity 3.8-fold, and the combination of 9-cis-RA and TZ
induced a 6-fold increase in activity (the right panel of
Fig. 6A
). The yeast colonies in the presence of RXR were approximately
3- to 4-fold larger and showed more distinct salmon pink color than
those in the absence of RXR on plates containing 9-cis-RA
(data not shown). The observation that only yeast cells expressing RXR
were able to survive suggested that RXR was necessary to bridge between
PPAR
fused to the GAL4-DNA binding domain to p120 fused to the
GAL4-activation domain (Fig. 6B
). These results clearly demonstrated
that p120 interacts with the RXR of PPAR
/RXR heterodimers, and that
9-cis-RA is required for this interaction.
 |
DISCUSSION
|
---|
The present study demonstrated that p120 interacts with
PPAR
/RXR heterodimers on PPREs in a 9-cis-RA-dependent
manner, and p120 functions as a coactivator of PPAR
and RXR
heterodimers. Several other coactivators that interact with various
nuclear receptors to enhance transcriptional activity have been
reported. Some of these belong to the histone acetyltransferase family
(33), while others bind to other histone acetyltransferase proteins
such as CBP/p300, which contribute to promoter activation by altering
or disrupting the repressive chromatin structure (34, 35). The
coactivators of PPARs/RXR, SRC-1, PBP, and PGC-1 have been cloned. It
is of interest that these molecules function as coactivators for
PPAR
/RXR by different mechanisms. SRC-1 and PBP interact with both
PPAR and RXR in a ligand-dependent manner, and the AF-2 region of PPARs
is important for their interactions (28, 30). A more recent study of
the crystal structure using x-ray analysis demonstrated that the AF-2
region of PPAR
formed a pocket-like structure and bound to SRC-1
(36). In fact, in our GST pull-down study the interaction domain of
SRC-1 clearly interacted with PPAR
in a ligand-independent manner
(Fig. 3
). On the other hand, PGC-1 has been reported to interact with
PPAR
in a ligand-independent manner, and the hinge region of PPAR
is essential for the interaction (29). In contrast to these
coactivators, the present results from EMSA and GST pull-down analyses
suggested that p120 did not interact with PPAR
at all but interacted
with RXR in a 9-cis-RA-dependent manner. These observations
indicated that the transactivation of ligand-induced PPAR
/RXR by
p120 is mediated through the RXR of PPAR
/RXR heterodimer complex. In
addition, the expression of the p120 gene in fat tissue indicated a
cooperative relationship between p120 and PPAR
in adipocyte
differentiation.
Although the yeast two-hybrid assay is a powerful technique with which
to analyze protein-protein interactions, this system is limited to
detection of protein interactions between two proteins. Thus, this
system fails to detect ternary complex formation. Since the
three-hybrid system is helpful for detection of X/Y/Z complex formation
when X does not bind to Z individually but only binds to the complex
produced by the combination of X/Y and Y/Z (37), we used this assay to
further confirm the interaction of p120 and RXR on RXR/PPAR
complex.
Our results clearly demonstrated the interaction of p120 (X) with the
PPAR
(Z) molecule only in the presence of RXR (Y). Furthermore, this
interaction required the presence of 9-cis-RA, and TZ did
not affect this protein-protein interaction (Fig. 7
). Taken together, these results
indicated that p120 act as a coactivator of PPAR/RXR through its
interaction with the RXR molecule. Furthermore, this yeast three-hybrid
assay is a useful strategy for detecting the partners of various
heterodimer complexes.
However, the reason for the indirect activation by p120 in the presence
of TZ alone in the CV-1 cells remains unclear. The first possibility is
that p120 interacts directly with other transcriptional factors, such
as CBP, which can interact with PPAR
in the presence of TZ (38).
This was supported by our previous results, which demonstrated
synergistic activation between p120 and CBP in a transfection study
using the TRß1 expression vector and TRE-TK Luc reporter vector. The
second possibility is that endogenous ligands for RXR, other than
9-cis RA, exist which might cooperate with exogenous
TZ in the serum used in the transfection experiments. The third
possibility is that not all retinoid is removed in stripped media and
that TZ then potentiates the interaction.
SRC-1 is the best characterized nuclear receptor coactivator. Although
SRC-1 was originally isolated as a protein interacting with PR, it
interacts with most known nuclear receptors, including TR, PPAR, RXR,
RAR, ER, AR, VitDR, and GR, and functions as a coactivator for these
receptors to enhance transcriptional activation (3). In contrast,
functional analysis of p120 revealed enhancement of the transcriptional
activation only by TR, PPAR, RXR, and AR, but not by ER or RAR.
Although it remains unclear whether p120 interacts with AR, ER, and
RAR, the present study demonstrated the importance of AA 186297 of
p120, containing the LXXLL motif, which is a signature sequence for the
binding of coactivators to nuclear receptors (39), for the interaction
with RXR and TR, but not with PPAR
. These findings indicated the
specificity of nuclear receptor-coactivator interactions and
suggested that the LXXLL motif is not sufficient for this
interaction. Therefore, it is speculated that the specific recruitment
of coactivators by nuclear receptors may allow the flexible regulation
by nuclear receptors in vivo. Further studies are required
to determine how these coactivators affect each other and regulate
ligand-dependent activation of gene transcription by nuclear
receptors.
 |
MATERIALS AND METHODS
|
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Plasmids and Constructs
The full-length mouse PPAR
2 cDNA was amplified by PCR using
the mouse skeletal muscle cDNA as a template (Marathon cDNA,
CLONTECH Laboratories, Inc.). The PCR product was verified
as PPAR
2 cDNA by sequencing and was cloned into the expression
vector pKCR2. For the yeast two-hybrid assay, the ligand-binding domain
of PPAR
2 (AA 193505) was subcloned into the pGBT9 plasmid
(CLONTECH Laboratories, Inc.). The human SRC-1 cDNA was
kindly provided by Dr. Takeshita (Harvard Medical School, Boston) (40).
For the GST pull-down assay, the interacting domain of SRC-1 (AA
583779) was generated by PCR amplification and inserted into the
pGEX-4T-1 plasmid (Amersham Pharmacia Biotech, Piscataway,
NJ). The PPRE reporter construct consisted of two copies of the DR1
element upstream of the TK109 promoter in the vector pA3Luc (26). TZ
was provided by Sankyo Co., Ltd. (Tokyo, Japan), and
9-cis-RA was purchased from Sigma Chemical Co.
(St. Louis, MO). These chemicals were dissolved in dimethyl
sulfoxide.
RT-PCR
Human mesenteric fat tissue was obtained at the time of
operation at the First Department of Surgery in Gunma University
Hospital (courtesy of Dr. Mochiki). Total RNA was extracted using the
modified acid-phenol method as described previously (40), and 2 µg of
total RNA were reverse transcribed into first-strand cDNA for 2 h
at 37 C using Moloney murine leukemia virus reverse transcriptase
(Roche Molecular Biochemicals, Indianapolis, IN). p120
cDNA and SRC-1 cDNA were amplified from 3 µl of cDNA (from a total of
20 µl) using the following primer combinations of sense/antisense
primers (p120: 5'-tcaaggtggaacctgca-3'/5'-tagcattgtgtatgctga-3':
nucleotide 1729 to 2235), (SRC-1:
5'-atggtgccgatgccaatccct-3'/5'-ttcagtcagtagctgctgaag-3':
nucleotide 3957 to 4514). PCR was carried out for 30 cycles consisting
of denaturation for 1 min at 94 C, annealing for 2 min at 60 C, and
extension for 2 min at 72 C, followed by a 15-min final extension at 72
C. Twenty microliters (50 µl total) of PCR products were visualizing
by agarose gel electrophoresis. The Southern blot analysis of PCR
products was performed. The PCR products were blotted onto Hybond
N+ membrane and hybridized with the p120 or SRC-1 cDNA
probes labeled with [
32P]dCTP using a DNA labeling kit
(Amersham Pharmacia Biotech).
Transfection
The CV-1 cells were maintained in DMEM containing 10% FCS, 0.25
mg/ml streptomycin, 100 mg/ml penicillin, and 0.25 mg/ml Amphotericin
(Life Technologies, Inc., Rockville, MD). The cells
were plated in six-well plates 24 h before transfection, and the
medium was changed 4 h before transfection. Using the
calcium-phosphate method, the cells were transfected with the following
amounts of DNA per six wells: 10 µg of pA3 Luc-TK reporter construct
containing two DR1 elements upstream; 100 ng of pKCR2-mouse PPAR
2;
100 ng of pKCR2-human RXR
; and 3 µg of either pKCR2-p120 or 3 µg
of pKCR2. Sixteen hours after transfection, the cells were cultured in
the absence or presence of 10 µM TZ or 1 µM
9-cis-RA for 24 h and then harvested for luciferase
assays after a further 24-h incubation. Luciferase activities were
normalized by protein content. All experiments were performed in
triplicate and repeated at least twice. The data shown are pooled
results ± SE.
GST Pull-Down Assay
Purified GST and GST fusion protein were bound to
glutathione-Sepharose beads. PPAR
2 and RXR
labeled with
[35S]methionine were synthesized by the TNT-coupled
in vitro translation system (Promega Corp.,
Madison, WI). These labeled proteins were incubated with GST-Sepharose
or GST-fusion proteins-Sepharose for 2 h at 4 C in the presence or
absence of the appropriate ligands (10 µM TZ or 1
µM 9-cis- RA) in reaction buffer (150
mM NaCl, 20 mM Tris, pH 7.5, 0.3% NP-40, 0.1
mM EDTA, 1 mM dithiothreitol, and 1
mM PMSF). The beads were then recovered by centrifugation.
After washing the beads, bound proteins were eluted in Laemmli buffer,
boiled for 2 min, and analyzed by SDS-PAGE followed by
autoradiography.
Yeast Two-Hybrid and Three-Hybrid Assays
p120 cDNA ligated into the plasmid pGAD24 expressing the GAL4
activating domain, the PPAR
cDNA encoding AA 193505 ligated to the
plasmid pGBT9 (42) expressing the GAL4-binding protein, or the
full-length hRXR
ligated to pGBT9 in frame, and pAUR123 (43)
expressing hRXR
protein, were cotransformed into yeast HF7c cells,
and transformants were plated onto synthetic complete medium plates
lacking histidine, leucine, and tryptophan. Liquid assays of
ß-galactosidase were carried out as described previously (1).
EMSA
The sequence,
5'-acgtagaagcttgaaatgAGGTAAAAGGTCAg-agtccaagct-3',
derived from the mouse leptin promoter region, was used as the DR1
probe (26). The probe was labeled by [
-32P]dCTP using
the PCR method. In vitro translated mPPAR
2, hRXR
proteins, and GST or GST-p120 AA187297 were incubated with 100,000
cpm of the labeled double-stranded oligonucleotide in the presence of 1
µg of poly(dI-dC), 1 mM dithiothreitol, 0.1 µg of
salmon sperm DNA, and EMSA binding buffer (20% glycerol, 20
mM HEPES, 50 mM KCl) was added to a final
volume of 30 µl. Reaction was performed at room temperature for 20
min, and the samples were analyzed on 5% nondenaturing polyacrylamide
gels. Electroporation was performed at 250 V for 90 min.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Takeshita for the SRC-1 plasmid and Dr. Mochiki for
the fat tissue specimen.
 |
FOOTNOTES
|
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Address requests for reprints to: Tsuyoshi Monden, M.D., First Department of Internal Medicine, Gunma University School of Medicine, 339-15 Showa-machi, Maebashi, Gunma 371-8511, Japan.
Received for publication February 19, 1999.
Revision received June 2, 1999.
Accepted for publication June 29, 1999.
 |
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