Interactions of ultraspiracle with ecdysone receptor in the
transduction of ecdysone- and juvenile hormone-signaling
Fang Fang
2
, Yong Xu
1
, Davy Jones
2
and Grace Jones
1
1 Department of Biology, University of Kentucky, Lexington, KY, USA
2 The Graduate Center for Toxicology, University of Kentucky, Lexington, KY, USA
Keywords
ecdysone receptor; juvenile hormone;
methyl epoxyfarnesoate; retinoid-x-receptor;
ultraspiracle
Correspondence
G. Jones, Department of Biology, University
of Kentuckey, 304 Morgan Building,
Lexington, KY 40506, USA
Fax: +1 859 257 7505
Tel: +1 859 257 2105
E-mail: [email protected]
(Received 14 September 2004, revised 13
January 2005, accepted 21 January 2005)
doi:10.1111/j.1742-4658.2005.04578.x
Analyses of integration of two-hormone signaling through the vertebrate
nuclear hormone receptors, for which the retinoid X receptor is one part-
ner, have generated a number of mechanistic models, including those des-
cribed as ‘subordination’ models wherein ligand-activation of one partner
is subordinate to the liganded state of the other partner. However, mecha-

20OHE, 20-OH ecdysone; DR1 and IR1, direct repeat and inverted repeat with one intervening base between repeats, respectively; EcR,
ecdysone receptor; EMSA, electrophoretic mobility shift assay; JH, juvenile hormone; JHECore, core promoter from juvenile hormone
esterase gene; RA, retinoic acid; RXR, retinoid-X-receptor; USP, ultraspiracle.
FEBS Journal 272 (2005) 1577–1589 ª 2005 FEBS 1577
Nuclear hormone receptors play key roles in metazoan
development, metabolic homeostasis, and response to
xenobiotics. Some of these nuclear receptors bind lig-
ands that modulate the regulatory effect of these recep-
tors on gene transcription, while other receptors are
apparently constituitively active and unregulated by
dynamic equilibrium with ligand [1]. For example, the
retinoic acid receptor (RAR) responds to signaling by
all-trans retinoic acid (at-RA) and 9-cis RA, in regula-
tion of fetal limb development [2]. The receptor FXR
binds to catabolites in regulation of sterol pathways,
the receptor PXR is activated by binding to certain
xenobiotic compounds, while transcription-enhancing
activity of the receptor CAR is suppressed by binding
to xenobiotic compounds [3].
Each of the above ligand-binding receptors and their
close relatives functions as a heterodimer with the
(also homodimer-forming) retinoid-X-receptor (RXR),
which itself can be activated by the RAR ligand 9-cis
RA [4]. The occurrence of such multiple partner recep-
tor complexes, and their corresponding multiple lig-
ands, raises questions about the integration of multiple
ligand signaling through the same receptor complex.
There is some controversy in the nuclear receptor field
as to whether RXR, the vertebrate ortholog of USP,
can independently bind ligand when RXR is in com-

Thus far, essentially all of such studies on integra-
tion of hormone signaling using cloned nuclear recep-
tors have involved vertebrate receptors. However, one
of the most dramatic examples of integrated signaling
by lipoidal regulators is insect metamorphosis, which
is regulated by the interplay between the steroid 20-
OH ecdysone (20OHE) and the terpenoid methyl
epoxyfarnesoate [juvenile hormone III, (JHIII)]. The
20OHE receptor (EcR) has been isolated for over a
decade [15], and was determined to function in vivo in
heterodimer with ultraspiracle (USP), an ortholog of
RXR [16]. Most studies on the EcR as a ligand bind-
ing receptor have focussed EcR binding of its ligand
while USP is premised or utilized as a ligandless dimer
partner. More recently, our studies in a model Sf9 cell
transfection system utilizing a natural core promoter
of the JH-activated juvenile hormone esterase gene [17]
have established that USP can transduce transcrip-
tional activation by JH III by way of binding of JH III
or closely related structures to the ligand binding
pocket of USP [18]. This binding of JH III by USP
induces change in USP tertiary conformation [18] and
induces or stabilizes USP homodimerization [19].
There has been renewed interest in how JH and
20-OH ecdysone signaling are integrated at a molecular
level [20,21]. Yet, thus far, there have been no reports
on the existence and nature of integration of JH III
and 20OHE signaling through the molecules of the
EcR ⁄ USP heterodimer complex. In this study, we used
cloned EcR and USP to assess the relationship of the

(0.1–30 lm) in the presence of 1 lm 20-OHE or the
dose range of JH III alone, and the activation ratio in
relation to the solvent (EtOH) treatment calculated. As
shown in Fig. 2, when the data for activation by
JH III alone vs. JH III plus 20-OHE were plotted
for each promoter construct, the resultant slopes of
the plots are not the same. The shallower slope for the
IR1JHECore promoter construct compared to the
DR1bJHE construct shows that the IR1JHECore con-
struct transduced a greater effect of JH III on 20OHE
than occurred with the DR1JHECore construct. For
the IR1JHECore, JH III alone at 30 lm yielded an
approximately twofold induction, 20OHE alone at
1 lm yielded an approximately 35-fold induction, but
together the two hormones yielded a 45-fold induction,
which is distinctly greater than an additive effect.
These results show that the transcriptional interaction
of these two hormones is transduced through the hor-
mone response element, and not through some other
region of the transfected plasmids. Functionally
important is that this result shows that the opposite
orientation of the second half site of what is otherwise
an identical hormone response element causes the two
response elements to differ in their effectiveness to pro-
mote this interaction between JH III and 20-OHE.
USP Interaction with DNA and EcR
We have demonstrated previously that under our con-
ditions, purified dUSP can bind to the DR12 motif
[19]. Using similar conditions, we confirmed by elec-
trophoretic mobility shift assay (EMSA) and supershift

flatter slope, yielded a much stronger effect of adding JHIII to
20OHE than was exhibited through the DR1 motif.
F. Fang et al. Interaction of ultraspiracle and ecdysone receptor in hormone signaling
FEBS Journal 272 (2005) 1577–1589 ª 2005 FEBS 1579
complex is observed (Fig. 3B). The specificity of the
binding is again confirmed by the competition of unla-
belled DR1 probe. The presence of USP in the com-
plex was confirmed by a supershift formed upon the
addition of a monoclonal antibody (AB11) specific for
USP (arrow). The specificity of the supershift was con-
firmed by the absence of a supershift when a monoclo-
nal antibody to an unrelated antigen (ELAV) was
used. These results confirm that USP binds to the
same DR1 hormone response motif that is used in the
DR1JHECore transfection construct.
We also used this system to test for the binding of
EcR to the same DR1 hormone response element. As
the monoclonal antibody to the Drosophila ecdysone
receptor (dEcR) does not cross-react well with the
endogenous Sf9 ecdysone receptor, we transfected Sf9
cells with a plasmid expressing dEcR, and harvested
the cells for binding of the extract to the DR1 probe.
As shown in Fig. 3C, a specific complex was observed
binding to the probe, which could be competed by
unlabelled probe (self) DNA, but not by an unrelated
(nonself) DNA. The complex could be supershifted by
the monoclonal antibody to dEcR (arrow), establishing
that EcR expressed in Sf9 cells can bind to the DR1
motif.
The direct interaction of USP with EcR in the Sf9

was compared at the same time to the dose depend-
ence of JH III action to increase the transcriptional
induction of 1 lm 20OHE. As shown in Fig. 4A, the
region of the dose-dependent JH III action to increase
AB C D
Fig. 3. Binding of ultraspiracle (USP) to DR1 motif and to ecdysone receptor (EcR). (A) Electrophoretic mobility shift assay (EMSA) of recom-
binant dUSP binding to DR1 probe (left lane), and supershift (arrow) with AB11anti-dUSP monoclonal Ig (middle lane). The negative control
shows no supershift with monoclonal antibody against irrelevant ELAV protein (right lane). (B) EMSA with DR1 probe using nuclear extract
from Sf9 cells. The major shift-band is specific on account of its competition with self but not with nonself unlabelled competitor. The com-
plex on the DR1 probe contains USP, as seen by the supershift (arrow) with the AB11 anti-USP monoclonal Ig. (C) EMSA with DR1 probe
using nuclear extract from Sf9 cells transfected with expression plasmid for dEcR. The major shift-band (leftmost lane) is specific on account
of its competition with self but not with nonself unlabelled competitor. The complex on the DR1 probe contains dEcR, as seen by the super-
shift (arrow) with the antidEcR monoclonal antibody. (D) USP binds with EcR in Sf9 nuclear extracts. Lysates from Sf9 cells cotransfected
with plasmids expressing dUSP, or dEcR, or both, or empty expression vector, were first immunoprecipitated with AB11 anti-dUSP mAb,
and the pellet subjected to immunoblotting (following SDS ⁄ PAGE), using anti-dEcR mAb.
Interaction of ultraspiracle and ecdysone receptor in hormone signaling F. Fang et al.
1580 FEBS Journal 272 (2005) 1577–1589 ª 2005 FEBS
the transcriptional activation by 20OHE closely paral-
lels the mid-micromolar region of the action of JH III
alone to induce transcription. These results suggest
that the mechanism of synergism is not one in which
the presence of 20OHE lowers the concentration of
JH III at which JH III exerts its transcriptional action.
These results also are also evidence that the site of
JH III action for its effect to alone induce transcrip-
tion of the DR1JHECore is the same as, or has a titra-
tion curve indistinguishable in this transfection assay
from, the site of JH III action for its transcriptional
interaction with 20OHE.
Our previous studies have demonstrated that JH III

factor that is needed for 20OHE activation, but not
JH III-activation, is already limiting before the over-
expression of more exogenous USP.
Fig. 4. Effect of treatment with hormones, and of receptor expression, on activation of the DR1JHECore reporter promoter. (A) Activation of
the DR1JHECore promoter by JH III alone (black histogram bars) or together with 1 l
M 20-OHE (white histogram bars). The values for JH III
alone are plotted at 20· their actual value, to enable more direct visualization that the range of JH III action alone is over a similar range of
its action in the presence of 20OHE. These data are from a single experiment. (B) Differential effect of transfection of increasing concentra-
tions of dUSP-expressing plasmid on action of JH III alone (black histogram bars) or 20OHE alone (white histogram bars) to activate
DR1JHECore reporter promoter. Data are average (±SE) of three independent experiments. (C) Transfection of increasing concentrations of
dUSP-expressing plasmid, while increasing the transcriptional activation arising from treatment with JH III alone (d), does not result in
enhancement of 20OHE-activation by JH III (s). Data points are the average of two independent experiments. (D) Effect of transfection of
increasing concentrations of dEcR-expressing plasmid on action of JH III alone (black histogram bars) or 20OHE alone (white histogram bars)
to activate DR1JHECore reporter promoter. Data are average (± SE) of two independent experiments.
F. Fang et al. Interaction of ultraspiracle and ecdysone receptor in hormone signaling
FEBS Journal 272 (2005) 1577–1589 ª 2005 FEBS 1581
We then assessed the effect of overexpression of
dUSP on the transcriptional activation pathways that
are induced by 20OHE alone and on the interaction of
JH III and 20OHE. In order to visualize more clearly
the effects, the data were analyzed for the fold change
in the hormonal activation that was caused by the
transfection of a particular amount of USP-expressing
plasmid. So, for example, the value of 2.0· for
1000 ng of wtdUSP-expressing plasmid means that the
JH III induction was yet another 2· higher than the
induction already caused by JH III in the absence of
transfected dUSP. As shown in Fig. 4C, as more USP-
expressing plasmid was transfected, there was an
increasingly greater transcriptional activation by

(but which does not aid 20OHE activation), dUSP
oligomer (we interpret this to include homodimer)
exists in Sf9 extracts.
Is EcR part of the JH III-activation pathway?
The indication that overexpressed USP does not aid
the 20OHE-activation pathway prompted us to exam-
Fig. 5. Immunoprecipitation of USP homodimer. Sf9 cells were transfected with the indicated expression plasmids. (A) Aliquots from total
cell lysates were loaded directly to SDS ⁄ PAGE for immunoblotting with the indicated antibody, which confirmed that the GFP-dUSP and
HA-dUSP were expressed in the cells transfected with the respective expression plasmid, and as a negative control neither was detected in
cells transfected with the empty expression plasmid. The upper blot in panel A shows the reactive band for GFP-dUSP present in cells trans-
fected with GFP-dUSP-expressing plasmid and not in cells transfected with empty vector. The lower blot in (A) shows the reactive band for
HA-dUSP present in cells transfected with HA-dUSP-expressing plasmid and not in cells transfected with empty vector. (B) Lysates from
cells transfected with the indicated plasmid constructs were first immunoprecipitated with anti-HA Ig, and the immunoprecipitate then sub-
jected to immunoblotting and probing with anti-GFP Ig. The only treatment to yield a 101 kDa corresponding to GFP-dUSP was that for cells
cotransfected with both the plasmids encoding GFP-dUSP and HA-dUSP. This result shows the presence of USP homodimer in the cell
lysates.
Interaction of ultraspiracle and ecdysone receptor in hormone signaling F. Fang et al.
1582 FEBS Journal 272 (2005) 1577–1589 ª 2005 FEBS
ine a reciprocal question: whether EcR is participa-
ting in the JH III alone-activation pathway. As one
approach to this question, we tested the effect of over-
expression of dEcR on the JH III activation pathway.
The pattern observed when dEcR was overexpressed
was the opposite of that observed for the case when
dUSP was overexpressed. As shown in Fig. 4D, as the
amount of plasmid overexpressing EcR was increased
progressively, the fold induction caused by treatment
with 20OHE alone also increased progressively. How-
ever, at the same time, there was no increase in the
level of induction caused by treatment with JH III

a dominant negative in the Sf9 cell transfection,
JH III-activation pathway. As shown in Fig. 6, addi-
tion of JH III to Sf9 cells transfected with the
DR1JHECore plasmid resulted in an induction of
promoter activity. However, cotransfection with pro-
gressively greater amounts of the mutant L314R
dUSP-expressing plasmid caused a progressive decrease
in the JH III-inducibility of the DR1JHECore promo-
ter (Fig. 6, upper panel). To further test whether the
JH III activation pathway requires the presence of the
wild-type L314 USP, cells were transfected with a
dominant-negative acting dose of L314R-expressing
plasmid (that suppressed JH III-activation), but were
also cotransfected with increasing amounts of plasmid
expressing wtUSP. The outcome was that the increas-
ing dose of wtUSP progressively rescued the JH III-
activation of the DR1bJHECore promoter (Fig. 6,
lower panel). These results further confirmed the parti-
cipation of wtUSP in the JH III-activation pathway,
and in particular establish that the wild-type confor-
mation of the surface near L314 is necessary for USP
transduction of JH III-activation.
Discussion
Juvenile hormone transcriptional activation
through USP
An important area of investigation in the mechanisms
of invertebrate hormone action is the identification of
Fig. 6. Role of JH III-activated transcription by hydrophobic residue
(L314) in putative coactivator-binding hydrophobic groove of USP.
(A) Activity of DR1JHECore promoter in Sf9 cells cotransfected

The physiological integration of juvenile hormone and
20OHE signaling has for several decades been an
underpinning of models for regulation of the complex
developmental transition of insect metamorphosis
[26,27], but the molecular mechanisms by which that
integration of signaling may be accomplished has
been frustratingly elusive [28,29]. USP does not bind
20OHE [30–32], and in fact EcR is the only inverteb-
rate nuclear hormone receptor shown to undergo
direct transcriptional activation by 20OHE [33]. It is
thus unlikely that the integration of nuclear JH III
and 20OHE signaling is mediated directly by a USP
homodimer, or by a USP heterodimer with another
partner other than EcR. Przibilla et al. [34] have iden-
tified mutations to USP residues that exert allosteric
effects on the activation of the EcR⁄ USP complex by
20-OHE alone, but the operation of JH on that com-
plex was not investigated. Recently, Kethidi et al. [35]
have reported a regulatory element through which JH
signaling suppresses ecdysone activation, but the com-
ponents of the complex binding at the element were
not ascertained. Also recently, Dubrovsky et al. [21]
have identified a specific target gene (E75A) for which
ecdysone activation is synergized by JH, but the
direct site of JH action in that synergism was not
reported.
In this study, we have demonstrated the enhanced
activation of the JHECore reporter promoter by
cotreatment with JH III (or its metabolite in cultured
Sf9 cells) and 20OHE. This action is mediated through

transduction of 20OHE-signaling through the EcR
component of the EcR ⁄ USP heterodimer, prompts a
hypothesis that the enhancement conferred by JH III,
when cells are cotreated with JH III and 20OHE, is
through the USP component of the EcR ⁄ USP het-
erodimer. Consistent with that hypothesis is our
observation that the dose-dependence of JH III action
(alone) to activate the DR1JHECore was in the same
dose range as its effect on transcriptional activation
together with 20OHE. This result indicates that the
site of JH III action for activation of DR1JHECore
(i.e, USP) could be the same site as is involved in the
JH III synergism of 20OHE (i.e. the USP component
of EcR ⁄ USP). Also consistent with that model, we
observed that under conditions of EcR overexpression,
increased 20OHE activation, there exists an EcR ⁄ USP
complex in nuclear extracts that can bind to the
DR1 enhancer, rendering the USP partner available
at the DR1 motif to integrate the JH III-signaling.
We therefore postulate that during the integra-
tion of action of JH III and 20OHE to activate
DR1JHECore transcription in Sf9 cells, the site of
Interaction of ultraspiracle and ecdysone receptor in hormone signaling F. Fang et al.
1584 FEBS Journal 272 (2005) 1577–1589 ª 2005 FEBS
JH III action is the USP component of the USP ⁄ EcR
heterodimer.
Our recent experiments show that transgenic dUSP,
mutated in the ligand pocket for reduced JH III bind-
ing, is unable to rescue Drosophila melanogaster (null
for USP) from a lethal period from pupariation to

EcR, leading to increased formation of EcR ⁄ USP het-
erodimer, did not yield increased cellular response to
treatment with JH III alone, even though USP is pre-
sent as a potential JH III target in the EcR ⁄ USP het-
erodimer. There has been considerable controversy in
the vertebrate nuclear receptor field concerning the
mechanistic context of ‘subordination’ of 9-cis RA
signaling through RXR in relation to the particular
RXR heterodimer partner and the liganded status of
that partner. Some reports have indicated that activa-
tion of RXR by ligand is not permitted by the
heterodimer partner thyroid hormone receptor (VDR),
and thus is a ‘subordinate’ partner to VDR [6]. Other
studies find that RXR in the RXR ⁄ TR heterodimer
can bind ligand but just not dissociate corepressor
bound to RXR [39], while yet other investigators
adduce the RXR subunit binds ligand but with the
effect to dislodge corepressor from the TR subunit
[40]. The inability of the RXR partner to respond
in vivo to RXR ligand when partnered to RAR has
also been taken as evidence that the ligand-dependent
activity of RXR is ’subordinated’ to that of RAR
[41,42]. Evidence has been presented showing the ‘sub-
ordination’ in vivo of RXR to the liganded state of
RAR arises from the inability of RXR ligand to effect
dislodging of corepressor [10]. In the RXR ⁄ VDR3 sys-
tem, RXR has been modeled as a nonligand-binding
and therefore silent partner, but a recent report finds
that liganded VDR allosterically modifies the apo-RXR
from an unliganded conformation to a liganded-like

of the USP response to JH III is operating in the
presence of an unliganded EcR heterodimer partner.
Thus, our study has offered the first invertebrate
model system in which the subordination relationships
can be tested for two identified nuclear receptors for
which an activating ligand is available for each.
F. Fang et al. Interaction of ultraspiracle and ecdysone receptor in hormone signaling
FEBS Journal 272 (2005) 1577–1589 ª 2005 FEBS 1585
Juvenile hormone transcriptional activation
requires a specific USP surface feature
Our present study found that the transcriptional acti-
vation by USP in response to JH III requires the pres-
ence of wild-type amino acid sequence at the receptor
surface corresponding to the coactivator binding site in
the ortholog RXR. In the model vertebrate receptors,
this hydrophobic groove that serves as the binding site
of coactivators when the binding of ligand causes the
a-helix 12 to become repositioned to one edge of this
hydrophobic groove. When USP is concentrated to
10 mgÆmL
)1
(orders of magnitude above physiological
levels) and crystallized with a stabilizing fortuitous
pseudoligand (phospholipid), its a-helix 12 is observed
in an antagonist position covering this groove [46].
Our studies with USP prepared at 200· lower concen-
tration (much closer to physiological levels) have
shown that binding of JH III causes the a-helix 12 to
move in relative position [18]. In this study, we have
observed that the USP mutation L314R converts USP

munoprecipitation experiments, a hemagluttanin (HA)-tag
was placed at the N-terminus of the dUSP by cloning a
double-stranded oligomer of the following sequence (upper
strand, 5¢-AGCTACCCATACGACGTGCCAGACTACG
CATCTCTG-3¢) into the BamHI site of the above pIE1-4
vector already containing the dUSP coding sequence in the
PmeI ⁄ NotI sites. The dUSP mutant L314R was made from
the above wild-type dUSP in pIE1-4, by a QuikChange XL
Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA,
USA) where the sequence of the mutation-target primer
was: 5¢-CGACCAGGTGATTCTGagTGAAAGCCGCTT
GGATCG-3¢. Antisense dEcR in pIE1-4 was prepared by
cloning its PCR product with a reverse orientation in the
PmeI ⁄ NotI sites of the vector. All constructs were con-
firmed by sequencing. Expression of each receptor in trans-
fected Sf9 cells was confirmed by immunoblotting with
AB11 monoclonal antibody against dUSP (a gift from
F. Kafatos, European Molecular Biology Laboratory, Hei-
delberg, Germany), with monoclonal antibody against the
ecdysone receptor (a gift from R. Evans, Salk Institute,
La Jolla, CA, USA), with rabbit polyclonal antibody
against HA-tag (Abcam Ltd, Cambridge, UK), or with
monoclonal antibody against GFP (Chemicon Interna-
tional, Temecula, CA, USA).
The DR1JHECore promoter reporter construct in pGL3
luciferase reporter vector (Promega, Madison, WI, USA)
was prepared as described in Xu et al. [18]. First, the JHE-
Core promoter ()61 to +28 [17]); was subcloned into the
KpnI ⁄ BglII sites of this reporter vector. An NheI site was
then manufactured immediately 5¢- to the KpnI site. Com-

Spodoptera frugiperda cell line, Sf9, was maintained and
transfected as described previously [19]. To study the
role of USP in activation of the reporter promoter in
ligand-treated cells, dUSP cDNA in pIE1-4 and its mutant
derivative (L314R) were cotransfected with the reporter
construct. At 36 h after transfection, the cells were treated
with the respective compound in ethanol solvent (0.1%
final ethanol concentration) or just ethanol solvent only, or
left as a no-treatment control. After 48 h of the treatment,
the cells were harvested and the activity of the luciferase
reporter was measured using a luciferase assay kit (Prome-
ga) in a multipurpose scintillation counter (Beckman, Full-
erton, CA, USA). b-galactosidase activity was measured
using chlorophenol red-b-d-galactopyranoside monosodium
(CPRG; Roche Molecular Biochemicals, Indianapolis, IN,
USA) as a colorimetric substrate. On each occasion that
the given cell transfection assay condition was tested, three
separate replications (wells) were included, and the given
cell transfection assay condition was performed on at least
two or three independent occasions (days). We observed, as
is commonly observed with such cell line experiments, that
the pattern of result for the given ligand was consistent,
although the absolute level of the corresponding reporter
enzyme activity varied from one occasion to the next in
relation to the vigor of the cell culture at the time. Thus,
unless otherwise indicated, for each experiment we show a
typical result of one of the multiple occasions that the
experiment was performed. Sf9 cells express endogenous
EcR and endogenous USP. Thus, transfection of plasmid
expressing dEcR and dUSP is assessing the effect of the

P,
and used in gel-shift assay to test for specific binding by
either purified recombinant dUSP, or by components of Sf9
nuclear extracts. Binding conditions for recombinant dUSP
were 1.0 lg of dUSP, 1 lg dIdC, 3 lLof5· binding buffer
(1·:10mm of Tris ⁄ HCl, pH 7.5, 33 mm KCl, 1 mm
MgCl
2
, 0.5 mm EDTA, 0.5 mm dithiothreitol, 4% glycerol),
10 fmoles of labeled probe, self or nonself competitor at
100· (nonself competitor ¼ pGL3 vector polylinker seq-
uence), and in some cases also mAb AB11 anti-dUSP, all
in a binding reaction volume of 20 lL. Similar conditions
were used for assays performed with Sf9 nuclear extracts,
except that 10 lg of nuclear extract were used and the dIdC
was titrated to an optimum, depending on the particular
nuclear extract preparation, and that in some assays there
was included a monoclonal antibody against dEcR, or a
monoclonal antibody against dUSP or a monoclonal
antibody against the unrelated protein ELAV [47]. The
antibody was added 30 min before addition of the probe.
After incubation at 4 °C, the binding reaction contents
were subjected to native polyacrylamide gel electrophoresis
(6% acrylamide). After electrophoresis the gel was dried,
exposed to X-ray film (Kodak, Rochester, NY, USA), and
the resulting image scanned into Adobe photoshop.
Acknowledgements
This research was supported, in part, by NIH
DK39197 and GM463713.
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