Divergent Pathways Regulate Ligand-Independent Activation of ER
in SK-N-BE Neuroblastoma and COS-1 Renal Carcinoma Cells
Cesare Patrone,
Elisabetta Gianazza,
Sabrina Santagati,
Paola Agrati and
Adriana Maggi
Centre Molecular Pharmacology Laboratory (C.P., S.S., P.A.,
A.M.) and Atherosclerosis Laboratory (E.G.) Institute of
Pharmacological Sciences University of Milan Milan, Italy
120133
 |
ABSTRACT
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The
-estrogen receptor (ER
) transcriptional
activity can be regulated either by binding to the cognate ligand or by
intracellular signaling pathways responsive to a variety of factors
acting through cell membrane receptors. Studies carried out in HeLa and
COS-1 cells demonstrated that the cross-coupling between estrogen and
growth factor receptors is mediated by p21ras and requires
phosphorylation of a specific serine residue (Ser 118 in the human
ER
and Ser 122 in mouse ER
) located in the ER
N-terminal
activation function 1 (AF-1). Likewise, in the SK-N-BE neuroblastoma
cell line p21ras is involved in the cross-coupling between
insulin and ER
receptors. However, in this cell line Ser 122 is not
necessary for insulin-dependent activation of unliganded ER
. In
addition, after insulin activation, the electrophoretic mobility
associated to serine hyperphosphorylation of ER
in SK-N-BE and in
COS-1 cells is different. Our study rules out the possibility of
tyrosine phosporylation in unliganded ER
activation by means of
transactivation studies of ER
tyrosine mutants and analysis of Tyr
phosphorylation immunoreactivity. The two cofactors for steroid
receptors RIP 140 and SRC-1 do not seem to be specifically involved in
the insulin-induced ER
transactivation. The present study
demonstrates the possibility of an alternative, cell-specific pathway
of cross-coupling between intracellular and membrane receptors, which
might be of importance for the understanding of the physiological
significance of this mode of activation in the nervous system.
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INTRODUCTION
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Estrogens represent an important class of hormones for the
maturation and functioning of several tissues including the nervous
system (1, 2, 3). Estrogen action is mediated by intracellular receptors
[named estrogen receptor (ER)
and ERß], members of a large
family of hormonally inducible transcription factors. After binding to
the cognate ligand, the ERs modulate the transcription rate of target
genes by means of protein-protein interactions affecting the stability
of the preinitiation complex (4, 5, 6, 7). With regard to the ER
, it is
known that these interactions are mediated by two transactivation
regions: AF-1 located in the ER
N terminus and AF-2 located in the
hormone-binding domain at the C terminus (8, 9). The two
transactivating regions may function independently, or cooperate,
depending on the target promoter and the presence of tissue-specific
factors (10, 11, 12).
Phosphorylation plays an important role in the modulation of steroid
hormone receptor functions, including receptor processing and
shuttling, DNA binding, and transcriptional activation (13, 14, 15, 16). In
general, this posttranslational modification occurs on serine residues
(16, 17, 18, 19, 20, 21), but tyrosine phosphorylation has also been reported (22, 23).
The finding that ER
can be phosphorylated by p21ras- or
cAMP-dependent pathways indicates that this posttranslational
modification may be involved in the cross-coupling between nuclear
receptors and other signal transduction pathways. Indeed, several
signaling pathways responsive to a variety of hormones, including
dopamine (24), epidermal growth factor (25), insulin, and insulin
growth factor-1 (IGF-I) (26, 27), have been shown to activate ER
in
a ligand-independent fashion. The mutation of the major phosphorylation
site (Ser 118 in human and Ser 122 in mouse ER
) proved that the
p21ras-dependent pathway involves AF-1 in fibroblast-like
COS-1 (28) and epithelial-like HeLa cells (29). Because of the distant
embryological origin of COS-1 and HeLa cells, it has been assumed that
p21ras-dependent activation occurs via an identical
mechanism in all cell types. However, our previous studies in a cell
line of neural origin (SK-N-BE neuroblastoma cells) proved AF-2 and not
AF-1 as essential for p21ras-dependent ER
activation
(30). This finding prompted us to further investigate the diversities
of ER
-independent activation in our cell system.
The present results show that not only is Ser 122 dispensable for
p21ras-activation of ER
in neuroblastoma cells, but also
the phosphorylation of ER
and its capability of interaction with
coactivators can differ in this cell line. Our present study therefore
suggests the existence of cell-specific events controlling unliganded
ER
activity.
 |
RESULTS
|
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Mutation of Serine 122 Suppresses p21ras-Dependent Activation of
ER
in COS-1 but not in SK-N-BE Cells
In human ER
, phosphorylation of Ser118 is important for the
transcriptional activity of AF-1 (18). This site was recently shown to
be a target for mitogen-activated protein kinases and essential for the
cross-coupling between epidermal growth factor, IGF-I, and ERs (28, 29). These findings are in contrast with the observation that deletions
encompassing the entire AF-1 domain of the mouse ER
did not affect
the capability of insulin/IGF-I to activate unliganded ER
in
neuroblastoma cells (30).
To examine the function of the Ser122 phosphorylation site (the mouse
counterpart of the human Ser118) on p21ras-dependent
activation of ER
in neuroblastoma, SK-N-BE cells were cotransfected
with the dominant positive mutant of p21ras [
p21(Leu61)Hras] in the presence of the native (wtER) or
Ser122-Ala mutated mouse ER
. The reporter plasmid used was
pVEREtkCAT. Monitoring of the chloramphenicol acetyltransferase (CAT)
immunoreactivity indicated that, in SK-N-BE cells, the
p21ras dominant positive mutant increased the
transcriptional activity of unliganded wt ER
about 4-fold above the
control levels. In these cells, the activated p21ras could
still increase ER
transcriptional activity despite the presence of
the Ser122 to Ala mutation (Fig. 1
, top). De facto, p21ras activated the
mutated receptor to an even greater extent (
8-fold) than the wild
type. Conversely, in COS-1 cells, the 2- to 3-fold
p21ras-dependent activation of ER
was completely
abolished by the presence of this amino acid substitution (Fig. 1
, bottom). In both cells lines, the constitutive, but not the
estrogen-dependent, transcriptional activity of the Ser122-Ala mutant
was considerably lower than to wtER (-57% and -73% in COS-1 and
SK-N-BE cells, respectively) proving that also in SK-N-BE cells the
integrity of this phosphorylation site is of importance for ER
transactivation functions.
Western Blot Analysis of Native and Activated ER
in COS-1 and
SK-N-BE Cells
ER
can be phosphorylated in several serine residues, and the
accumulation of this phosphate groups changes its electrophoretic
migration profile (19, 21). To determine whether insulin could
influence the phosphorylation state of ER
, we studied its migration
profile in COS-1 and SK-N-BE cells after 18 h treatment with 1
µM insulin. A band of 66- kDa molecular mass,
corresponding to the uterine ER
, was revealed by the anti-ER
monoclonal antibody (Ab) H222 in untreated COS-1 and SK-N-BE cells
stably transfected with ER
[SK-ER3 (31)] (Fig. 2
, lanes 1 and 4). In some, but not all,
of the experiments, bands at lower molecular mass could be detected by
the H222 Ab. These bands could be due to the presence of degradation
products. After insulin stimulation, ER
migrated differently in
COS-1 and SK-N-BE cells. In COS-1 cells, ER
migrated as a triplet
(the majority of the receptor still remained in the 66-kDa band; about
30% migrated in the two upper bands), while no changes in the banding
pattern were observed in SK-N-BE cells (Fig. 2
, lanes 2 and 5).
After treatment with estradiol, a triplet was recognized by the H222 Ab
(Fig. 2
, lane 6) in both cell lines (Fig. 2
, lane 3 and 6). As shown by
several authors, this effect is most likely due to the ER
hyperphosphorylation occurring after ligand activation (17, 21, 32). In
both cell lines, estradiol treatment produced a 4050% decrease of
ER
immunoreactivity due to autologous down-regulation (33).
Since we have previously demonstrated that p21ras is
essential in insulin-dependent activation of ER
in neuroblastoma
cells, these data support the finding of the previous transient
transfection experiments, suggesting that the molecular events induced
by insulin differ in the two cell lines studied. Insulin-dependent
hyperphosphorylation of ER
can be observed only in COS-1 cells.
However, SK-N-BE cells seem to be supplied with the whole enzymatic
apparatus required for ligand-induced ER
posttranslational
modifications, as demonstrated by the fact that estrogen treatment
induces changes of ER
electrophoretic mobility superimposable to
those observed in COS-1 cells.
Tyrosine Phosphorylation Is Not Involved in Insulin-Dependent
Activation of ER
in SK-N-BE Cells Stably Transfected with ER
ER
has been reported to be phosphorylated also on tyrosine. The
phosphorylated site in this case seems to be unique (23) and localized
in a sequence that is phylogenetically very well conserved (Tyr 537 in
human and Tyr 541 in mouse ER
).
To investigate whether the insulin-dependent activation of ER
involved this residue, we next compared the transcriptional activity of
native ER
with mutants in which two different amino acids had been
substituted for tyrosine 541 (Y541-A and Y541-F). The mutants were
screened using a pVEREtkLUC reporter (Fig. 3
). The substitution of tyrosine with
phenylalanine in position 541 did not affect the transactivation
induced by insulin (or ß-estradiol) suggesting that this residue is
not a target for insulin activity. Alanine substitution in position 541
significantly increased (+100%) the ER
constitutive transcriptional
activity, according to observations of other authors.
To rule out the implication of other tyrosine residues in insulin
activation of ER
, SK-N-BE cells stably transfected with ER
(SK-ER3 cells) were treated for 18 h with insulin,
17ß-estradiol, or the appropriate solvent, and then harvested and
lysed. Equal amounts of whole-cell extract proteins were resolved by
denaturing gel electrophoresis. After blotting, the band corresponding
to ER
was detected with the H222 anti-ER
monoclonal Ab (Fig. 4
, right). The membrane was
then stripped and reprobed with the selective 4G10 anti-phosphotyrosine
monoclonal Ab (Fig. 4
, left). As shown before,
estrogen-dependent hyperphosphorylation produced several forms of ER
migrating at apparent 6668 kDa. Insulin treatment neither changed the
migration profile of the receptor nor influenced the staining intensity
of the 66-kDa protein with the 4G10 Ab. The same experiment was
repeated with immunoprecipitated ER
. Also in this case, in
insulin-treated cells, the intensity of the 4G10 staining was not
significantly different from control cells (Fig. 5
). A further proof that the two
antibodies, H222 and 4G10, were both recognizing the ER
is given by
the experiment done in ER
-negative SK-N-BE cells in which both
antibodies failed to recognize any protein of 6668 kDa (Fig. 5
).

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Figure 4. Constitutive or Insulin- and Estradiol-Induced
Phosphorylation of ER Stably Transfected in SK-N-BE Cells (SK-ER3)
Whole cell extracts (200 µg) were resolved by SDS-PAGE and
transferred to a nitrocellulose membrane. The membrane was stained with
the H222 Ab to label the ER (right panel), stripped,
and reprobed with the 4G10 antiphosphotyrosine Ab (left
panel). See Materials and Methods for further
experimental details. The autoradiograms represent one of three
separate experiments, all providing superimposable results.
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It is of interest to point out that, in spite of the down-regulation of
ER
, due to the estradiol treatment, the intensity of the 4G10
staining in Fig. 4
does not change. This suggests that, after estrogen
activation, tyrosine residues of ER
are phosphorylated.
The observations that mutations of tyrosine 541 do not block
insulin-dependent activation of ER
and that tyrosine phosphorylation
of ER
is not modified by insulin support the conclusion that
tyrosine phosphorylation of ER
is not relevant for insulin-dependent
activation in SK-N-BE cells.
SRC-1 and RIP-140 Do Not Play a Significant Role in
Insulin-Dependent Activation of ER
in SK-N-BE Cells
Cofactors and coactivators play an important role in the
transcriptional activity of ER
. We therefore examined whether, in
SK-N-BE cells, the ligand-independent activation of ER
was modulated
by the presence of two factors, namely RIP 140 (34) and SRC-1 (35),
known to operate through AF-2. Figure 6
shows that RIP 140 at various concentrations did not exhibit any
facilitator effect on insulin-dependent transcriptional efficiency of
ER
. In agreement with reports from other cell lines, this factor
facilitates estrogen-dependent activation of ER
, and its presence at
high concentration influences the basal transcriptional activities of
ER
(34). SRC-1 showed a concentration-dependent effect on the
constitutive transcriptional activity of the unliganded ER
. The
presence of insulin did not further augment ER
activity (Fig. 7
). The effect of SRC-1 on ER
constitutive transcription makes it difficult to establish whether this
coactivator displays any facilitator effect on insulin action.

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Figure 6. RIP 140 Cotransfection Does Not Affect
Insulin-Dependent Activation of Unliganded ER
SK-N-BE cells were transfected as described in Materials and
Methods in the presence or absence of increasing concentrations
of a vector expressing RIP 140 (0.03, 0.3, and 3 ng). The
bars represent the average ± SD of
three independent experiments, each done in triplicate.
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Figure 7. SRC-1 Effect on ER Transcriptional Activity in
SK-N-BE Cells
SK-N-BE were transfected as in Fig. 5 . The concentrations of the
SRC-1-expressing plasmid were: 100, 200, and 600 ng.
Bars represent a single experiment done in triplicate.
The experiment was repeated five times even with a wider range of SRC-1
concentrations with superimposable results.
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DISCUSSION
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The main conclusion of the present study is that, in SK-N-BE
neuroblastoma cells, insulin-dependent ER
activation occurs through
a diverse mechanism from the cell lines so far studied. In fact, while
insulin/IGF-I receptors and ER
are cross-coupled via the
p21ras pathway in several cells lines, SK-N-BE included, the
mechanism of unliganded ER
activation in neuroblastoma cells seems
to differ downstream of p21ras. We provide two lines of
evidence to support this conclusion: 1) insulin activates
transcriptionally the ER
mutant S122A in SK-N-BE, but not in COS-1
cells (Fig. 1
); and 2) insulin gives rise to two different ER
electrophoretic migration profiles in COS-1 and SK-N-BE cells (Fig. 2
).
These results support our previous studies in SK-N-BE, demonstrating
that only the ER
C terminus AF-2 is crucial for unliganded ER
activation (30), and are in agreement with others (28, 29) proving the
importance of serine phosphorylation in AF-1 in COS-1 cells.
Our study rules out Ser122 and ER
N terminus as a target for insulin
activity; however, since other serines in the hinge region and in the C
terminus were described as susceptible to phosphorylation, at present
we can not exclude that, in SK-N-BE cells, insulin activates ER
via serine phosphorylation. On the other hand, an
involvement of tyrosine phosphorylation appears as unlikely on the
basis of our study with the mutants of Tyr541 (the well recognized
tyrosine phosphorylation site in AF-2) (23) (Fig. 3
) and of the
immunoenzymatic labeling of ER
using the antiphosphotyrosine Ab 4G10
(Figs. 4
and 5
).
Transcription activation of ER
, as well as of other intracellular
receptors, requires a series of events initiated by the dissociation of
the receptor from the inhibitory proteins [heat shock protein (HSP),
immunophilins, etc.]. The question arising is how insulin can activate
the receptor. Considering that the receptor might not be entirely
assembled with HSP and that, as recently suggested by Smith et
al. (39), the ER
-HSP complexes might continuously dissociate
and reassociate, we propose that the quota of free receptor could be
the target for insulin activation. Hence, insulin might induce
posttranslational modifications enabling ER
to better interact with
transcription coactivators or might induce the synthesis of specific
coactivators capable of modulating the unliganded receptor activity. Of
interest is our observation that, in SK-N-BE cells, SRC-1 activates
transcriptionally unliganded ER
. Because in other cell lines SRC-1
does not interact with unliganded ER
, our findings might suggest
that in SK-N-BE the receptor conformation, possibly due to the presence
of specific factors, is more ready to recognize SRC-1 or the
transcriptional machinery. However, at this stage, we cannot point to
any SK-N-BE-specific molecule determining this effect.
The peculiarity of the cell line here studied seems to be restricted to
insulin-dependent activation of ER
. In fact, in the case of estrogen
activation, the SK-N-BE ER
undergoes changes in the electrophoretic
mobility (Fig. 2
) similar to those described in other cell systems by
Pateranne and co-workers and Le Goff et al. (19, 32).
Furthermore, the results of the studies carried out with the Tyr 541
mutants (Fig. 3
) are in complete agreement with those of White and
associates (36) and Weis et al. (37). However it is
noteworthy that, by the use of antiphosphotyrosine antibodies, we prove
that estrogen-dependent activation of ER
causes an increased
immunoreactivity (Fig. 4
), suggesting the possibility of
phosphorylation in tyrosines other than Tyr 541.
In conclusion, in all the cell types tested so far, the cross-coupling
between ER
and other intracellular signaling pathways was
demonstrated to occur via the AF-1 site. The neuroblastoma
cell line SK-N-BE represents an exception to this rule. At present it
is difficult to demonstrate that the peculiarity of the neuroblastoma
cell line reflects a physiological requirement for ER
action in
neural cells. However, in view of the multiple roles of ER
in the
nervous system, both during brain development and later in the mature
brain, we propose that neural cells may utilize insulin activation of
AF-2 as a default pathway to ensure the transcription of AF-2-dependent
target genes even in the presence of low blood level of estrogens. For
instance, it could prove beneficial that neural growth in the
developing brain, or survival and plasticity in both developing and
mature brain, was under the control of a multiplicity of hormonal
signals funnelled through the ER. Supporting this view is the
observation that insulin and its receptor are present at high
concentration in the same brain areas in which ER
appears to be
localized (40).
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MATERIALS AND METHODS
|
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Reagents
Chemicals were purchased from Bracco (Milan, Italy) if not
otherwise specified. Plasticware was from Corning (distributed by Disa,
Milan, Italy). Tissue culture media from Sigma Chemical Corp. (Milan,
Italy) and GIBCO (Paisley, U.K.). FBS (from Poxoid, Milan, Italy;
distributed by Unipath, Milan, Italy). Insulin and 17ß-estradiol
purchased from Sigma were resuspended and stored as previously
described (30). The murine ER expression plasmids pMT2MOR, pMT2MOR
S122, Y541-A, and Y541-F (37) and pBRIP 140 (34), were from M. G.
Parker (London, U.K.); pVEREtkLUC, pVEREtkCAT, pCMVßgal, and
pBK-CMVSRC-1 (35) were provided by M. J. Tsai (Houston, TX); the
dominant positive mutant p21(Leu-61)H-ras (41) was supplied
by E. Martegani (Milan, Italy).
Cell Culture and Transient Transfection
SK-N-BE and COS-1 cells were grown in RPMI 1640, without phenol
red and supplemented with 10% charcoal-stripped serum, and transfected
in 1% serum by the calcium phosphate coprecipitation method, as
previously described (30). Generally, 50,000 cells were plated in
24-multiwell plates and grown for 24 h in 200 µl of medium. For
the transfection, growth medium was replaced with 400 µl of DMEM to
which 50 µl of the calcium phosphate/DNA suspension were added. In
the 50-µl suspension the DNA concentration was as follows: 0.2
ng/µl of the internal control pCMVßgal, 0.6 ng/µl of plasmids
pMT2MOR or MOR mutants, 1.2 ng/µl of the reporter plasmids and the
carrier pGEM3Z to a final DNA concentration of 25 ng/µl. In some
experiments, the above plasmids were cotransfected with expression
plasmids for RIP 140, SRC-1, and p21(Leu-61)H-ras or the
corresponding empty vectors. Details in the amounts used are reported
in the figure legends. The reporters were either pVEREtkCAT or
pVEREtkLUC. In transient transfections the hormonal treatments were
done by addition of the specified hormones in the medium after removal
of the cotransfection mixture. We used 1 nM 17ß-estradiol
and 1 µM insulin; the treatments were carried out for
24 h, after which the cells were harvested and extracts were
prepared for ß-gal, CAT, or luciferase assays as previously specified
(30). Luciferase activity was measured by integrating the luminescence
signal for 15 sec; experimental values are expressed as arbitrary
luminescence units. CAT content was measured by an immunoenzymatic
method (CAT ELISA kit, Boehringer Mannheim, Milan, Italy), and
luciferase experimental values were normalized on the protein
concentration of the extract (42) and the ß-galactosidase
activity.
Western Analysis of ER
Protein
ER
-containing extracts were prepared in which the cells were
resuspended (
8 x 106) in 200 µl of lysis buffer
[50 mM Tris-HCl, pH 8, at 20 C, 150 mM NaCl,
10% (vol/vol) glycerol, 1 mM EGTA, 1 mM
sodium-orthovanadate, 5 µM ZnCl2, 100
mM NaF, 1 mM phenylmethylsulfonylfluoride,
1.3% (wt/vol) sodium-deoxycholate, 1% (vol/vol) Triton-X100]. The
cell lysate was frozen and thawed twice in liquid nitrogen for 1
min/cycle and centrifuged at 100,000 x g for 15 min.
The supernatants were collected and the protein content was measured by
Bradfords method (42). Equal amounts of protein extract (100500
µg) from COS-1 and SK-N-BE cells were either immunoprecipitated with
the C542 monoclonal anti-ER
Ab (Stressgene Biotechnology Corp.,
Victoria, British Columbia, Canada) or directly loaded (100200 µg)
on SDS-PAGE 412% T polyacrylamide gradients cast with discontinuous
Laemli buffer and resolved using a Protean II apparatus at 500 V and 70
mA. ER
was immunoprecipitated from the supernatant of the whole-cell
extracts above described. Five hundred micrograms of each sample were
incubated overnight at 4 C with 5 µg of the ER
monoclonal Ab C542.
Protein G Sepharose (10 mg) (Pharmacia Biotech, Uppsala, Sweden),
prewashed in buffer A (50 mM Tris-HCl, pH 8, 5% albumin)
and resuspended in 125 µl of lysis buffer, was added to the samples
and incubated for 3 h at 4 C with rotation. The pellets, obtained
after centrifugation for 15 sec at 4 C at 12,000 x g,
were washed four times with 500 µl of lysis buffer and once with
distilled water. Before loading on the gel, the immunoprecipitated
proteins of each sample were eluted from Protein G Sepharose by boiling
the pellet for 10 min in 30 µl electrophoresis buffer containing 10
µl of Laemmli buffer 3x (200 mM Tris-HCl, pH 8, 30%
glycerol, 6% SDS, 15% ß-mercaptoethanol, and 0.075 bromophenol
blue) and 20 µl of lysis buffer. Color markers (6.5205 kDa, Sigma)
were used as migration reference. The run was continued for 45 min
after the exit of the tracking dye off the anodic edge of the slab.
Electroblotting was at 400 mA for 9 h at 4 C in a Bio-Rad tank
(Protean 228 II). After protein staining with Red Ponceau, 50-mm
strips, centered on the albumin standard, were cut from nitrocellulose
filters. The membrane was saturated with 5% (wt/vol) milk proteins in
Tris borate buffer containing 0.1% Tween 20. Using the anti-ER rat
monoclonal H222 (Abbott Laboratories, Rome, Italy) as primary Ab, the
secondary Ab was a rabbit antirat-IgG Ab coupled with horseradish
peroxidase, HRP (Vector Laboratories, Burlingame, Ca). Both antibodies
were used at 1:10,000 dilution. When specified, the membrane was
stripped free of the immunoreagent, tested for successful stripping,
and reprobed with an antiserum raised against phosphotyrosine (4G10,
UBI, distributed by DISA, Milan, Italy) at 1:8,000 dilution. In this
case, the secondary Ab was an antimouse IgG HRP-coupled Ab (Vector
Laboratories, Burlingame, CA). The zymogram for peroxidase was
developed with the ECL reagent (Amersham, Milan, Italy). The
Hyperfilm-MP (Amersham, Milan, Italy) was exposed to chemoluminescent
radiation for 5 min to reveal ER bands and 20 min when the 4G10 Ab was
used.
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ACKNOWLEDGMENTS
|
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We are grateful to Raffaella Barbieri and Monica Rebecchi for
their excellent secretarial and technical assistance, to Elisabetta
Vegeto and Paolo Ciana for helpful comments, and to E. Martegani, M.
Parker, M. J. Tsai, and Abbott Laboratories for providing us with
reagents critical for the present study.
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FOOTNOTES
|
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Address requests for reprints to: Adriana Maggi, Via Balzaretti 9, Milan Italy.
This work was supported by the European Economic Community (BIOMED
Programme Pl962286), the Italian Association for Cancer Research
(AIRC), Telethon Italy (E600), and the Istituto Superiore di
Sanità (93/J/T61) and National Research Council (Target Project
Biotechnologies).
Received for publication November 17, 1997.
Revision received January 27, 1998.
Accepted for publication February 11, 1998.
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