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J Biol Chem, Vol. 274, Issue 46, 32904-32908, November 12, 1999
From the Fanconi anemia (FA) is a genetic disorder
characterized by bone marrow failure, congenital abnormalities, cancer
susceptibility, and a marked cellular hypersensitivity to DNA
interstrand cross-linking agents, which correlates with a defect in
ability to repair this type of damage. We have previously identified an
approximately 230-kDa protein present in a nuclear protein complex in
normal human lymphoblastoid cells that is involved in repair of DNA
interstrand cross-links and shows reduced levels in FA-A cell nuclei.
The FANCA gene appears to play a role in the stability or expression of
this protein. We now show that p230 is a well known structural protein,
human Fanconi anemia (FA)1 is
a recessively transmitted genetic disorder characterized by bone marrow
failure, diverse congenital abnormalities, and an increased incidence
of cancer (1-3). One of the distinguishing characteristics of this
disease is the marked cellular hypersensitivity to interstrand
cross-linking agents, which correlates with a defect in ability to
repair damage produced by these agents (3-7). The etiopathogenesis of
FA remains unclear, however, despite the fact that genes for three of
its eight known complementation groups (FANCA,
FANCC, and FANCG) have been cloned and their
protein products examined (8-11). How the DNA repair defect is related
to the FA genes or gene products or to the clinical manifestations of
the disorder is unclear.
We have isolated a chromatin-associated protein complex from the nuclei
of normal human lymphoblastoid cell nuclei and have shown that this
complex is involved in the repair of DNA interstrand cross-links
(12-15). A number of the proteins involved in nucleotide excision
repair are present in this complex (16). In FA-A and FA-D cells there
is a defect in the ability of this complex to incise DNA containing
interstrand cross-links (13, 14). We have recently shown that there is
a deficiency in FA-A cells in the levels of an approximately 230-kDa
protein present in this nuclear complex and that the FANCA
gene plays a role in the stability or expression of this protein
(16).
We have now determined the identity of the 230-kDa protein and shown
that it is the structural protein Chromatin-associated Protein Extracts--
Normal human (GM 1989 and GM 3299) lymphoblastoid cell lines were obtained from the Coriell
Institute for Medical Research, Camden, NJ. FA-A (HSC 72 and HSC 99),
FA-B (HSC 230), FA-C (HSC 536), and FA-D (HSC 62) lymphoblastoid cell
lines were a gift from Dr. Manuel Buchwald (Hospital for Sick Children,
Toronto, Canada). FA-A lymphoblastoid cells (HSC 72) were stably
transduced with a retroviral vector expressing the FANCA
cDNA (HSC 72-17) (23). Cell lines were grown in suspension culture
in RPMI 1640 medium as described previously and routinely checked for
mycoplasma using an American Type Culture Collection polymerase chain
reaction-based mycoplasm detection kit (13). HeLa cells were obtained
from Cellex Biosciences, Inc., Minneapolis, MN. Cell nuclei were
isolated, and the chromatin-associated proteins were extracted from
them in a series of steps as described previously (13, 24).
Development and Purification of Monoclonal antibodies
(mAb)--
mAbs were developed, as described previously, against
proteins in the endonuclease complex, pI 4.6, which was isolated from chromatin-associated proteins in the nuclei of normal human
lymphoblastoid cells and recognizes and incises DNA containing
interstrand cross-links (12, 16). One of the mAbs developed was against
the 230-kDa protein. This mAb, which completely inhibited activity of
the protein complex on DNA containing interstrand cross-links produced by 8-methoxypsoralen plus UVA light, was of the IgM class; it was
subsequently purified on a Superose 6HR 10/30 size exclusion column
(Amersham Pharmacia Biotech).
Identification of the 230-kDa Protein--
HeLa
chromatin-associated proteins were separated by SDS-PAGE on a 7-9.5%
gradient gel (15 × 20-cm). After separation, a portion of the gel
was cut off and electroblotted onto a nitrocellulose membrane. One-half
of the membrane was stained with colloidal gold (Bio-Rad), and the
other half was immunoblotted with anti-p230. In this way the 230-kDa
band could be identified on the stained membrane. The remainder of the
gel was stained with Coomassie, and the 230-kDa band was identified,
excised, and sent to the HHMI Biopolymer Facility/W. M. Keck
Foundation Biotechnology Resource Laboratory at Yale University for
mass spectrometric analysis by matrix-assisted laser desorption
ionization mass spectrometry (MALDI-MS).
At the Keck Facility, the protein was digested within the gel slice
with trypsin. The resulting digest was mixed with Staining of Electrophoretically Separated HeLa Cell
Proteins--
To get a better idea of the relative abundance of the
230-kDa protein in the HeLa cell chromatin-associated protein extracts, these extracts were separated by SDS-PAGE according to the method of
Laemmli (26). Samples were electrophoresed on two different 15 × 20-cm gels, each with a 4% stacking gel: one using a 7-9.5% gradient
(to maximize separation of the higher molecular weight range proteins),
and the other using a 12.5-15% gradient (for separation of the lower
molecular weight proteins). In addition, human erythroid spectrin
(Sigma) was also electrophoresed on the 7-9.5% gradient gel. Two
different molecular weight markers were run alongside these lanes:
NOVEX See Blue Ladder (4-250-kDa range) and Bio-Rad Kaleidoscope
Ladder (7.1-208-kDa range). After electrophoresis, gels were stained
with SYPRO orange (1:5,000 dilution) (Bio-Rad) in 10% acetic acid for
45 min to 1.5 h. Gels were visualized on a VISTA FluorimagerSI
using ImageQuant software (Molecular Dynamics).
Immunoprecipitation--
For immunoprecipitation (IP),
chromatin-associated protein extracts from normal human, HeLa, FA-A,
and transduced FA-A cells were prepared in 50 mM potassium
phosphate, pH 7.0, 1 mM EDTA, 1 mM
dithiothreitol, and 40% glycerol. For anti-FANCA IP, affinity-purified rabbit polyclonal antisera generated from the carboxyl-terminal region
of the FANCA protein, or pre-immune serum, was bound to protein
A-coated agarose beads (Sigma) in binding buffer (25 mM Tris-Cl, pH 7.3, 150 mM NaCl, 1% Triton X-100 plus
protease inhibitor mixture (Roche Molecular Biochemicals)). For
anti- Immunoblotting--
For immunoblotting, chromatin-associated
proteins from the nucleus of either normal human lymphoblastoid, HeLa,
FA-A, FA-B, FA-C, FA-D, or transduced FA-A cells, or human erythroid
spectrin (Sigma) were separated on SDS-PAGE, using either 7.5 × 8.5-cm or 15 × 20-cm gels, following the method of Laemmli (26).
The proteins were then transferred to nitrocellulose in transfer buffer (25 mM Tris-Cl, pH 8.3, 192 mM glycine, and
20% (v/v) methanol) at 100 volts for 2 or 2.5 h at 4 °C. The
membranes were blocked in blocking buffer for 1 h at room
temperature or overnight at 4 °C and then incubated in primary
antibody in TTBS overnight at 4 °C. After washing in TTBS, the blots
were incubated in secondary antibody (anti-mouse IgM, anti-mouse IgG,
or anti-rabbit IgG) or protein A conjugated to horseradish peroxidase
in 1% nonfat dried milk blocker (Bio-Rad) in TTBS. The blots were
washed further in TTBS and reacted with substrate (Pierce Ultra or
Pierce Super chemiluminescent substrate) and exposed to x-ray film
(Pierce). The primary antibodies used were anti- A mAb developed against the 230-kDa protein has been used in its
identification. This protein, after separation by gel electrophoresis and identification by Western blot analysis, was digested with trypsin
and analyzed by MALDI-MS. The rationale for using this approach is that
it is very sensitive and the mass spectrum of the peptide mixture,
resulting from the enzymatic digestion of the protein, provides a
fingerprint that can be specific enough to identify the protein
(27).
Using this method, the 230-kDa protein was identified as human
nonerythroid Confirmation of the 230-kDa protein as Electrophoretic separation of the proteins in the HeLa cell
chromatin-associated protein extracts showed that
Human 
Spectrin II and the Fanconi Anemia Proteins FANCA and
FANCC Interact to Form a Nuclear Complex*
§,§
Department of Pathology and Laboratory
Medicine, University of Medicine and Dentistry-New Jersey Medical
School, Newark, New Jersey 07103, § Graduate School of
Biomedical Sciences, Newark, New Jersey 07103, and ¶ University of
North Carolina Gene Therapy Center, University of North Carolina,
Chapel Hill, North Carolina 27599
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
spectrin II (SpII
*), and that levels of SpII* are
not only significantly reduced in FA-A cells but also in FA-B, FA-C and
FA-D cells (i.e. in all FA cell lines tested), suggesting a
role for these FA proteins in the stability or expression of SpII*. These studies also show that SpII* forms a complex in the nucleus with the FANCA and FANCC proteins. SpII* may thus
act as a scaffold to align or enhance interactions between FA proteins
and proteins involved in DNA repair. These results suggest that FA
represents a disorder in which there is a deficiency in
SpII*.
INTRODUCTION
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
spectrin II (SpII*). We
have also shown that levels of SpII* are significantly reduced in
FA-B, FA-C, and FA-D, as well as FA-A cells and that this protein forms
a complex in the nucleus with the FANCA and FANCC proteins. Thus
SpII* may act as a scaffold to align or enhance interactions between the FA proteins and other proteins in the nucleus such as those
involved in DNA repair. Because nonerythroid spectrin has been
shown to interact with proteins involved in a number of cellular
processes, such as DNA synthesis, cell cycle progression, gene
expression, signal transduction, and cell growth and differentiation (17-22), a deficiency in this protein in FA cells could have far reaching consequences due to the number of systems affected. This may
explain some of the diverse cellular and clinical defects that have
been reported in FA (1-3).
EXPERIMENTAL PROCEDURES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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-cyano-4-hydroxy cinnamic acid matrix solution and spotted onto a sample target (25).
The sample target was then introduced into the mass spectrometer, a
Micromass TofSpec S.E. that can be used in either the linear or
reflectron mode and is equipped with delayed extraction, which increases resolution and mass accuracy. The resulting peptide masses
were then subjected to peptide mass searching using Peptide Search and
Profound to identify proteins whose sequences were in the EMBL
nonredundant and OWL data bases, respectively.
spectrin IPs, anti- spectrin (prepared against chicken
blood cell membranes, specific for mammalian nonerythroid spectrin
and chicken spectrin) (Chemicon, mAb 1622), or anti-mouse
IgG1 (Sigma) (used as a control) was bound to protein
G-coated agarose beads (Sigma) in binding buffer. These beads were
blocked in blocking buffer (4% nonfat dried milk blocker (Bio-Rad) in
TTBS (25 mM Tris-Cl, pH 7.3, 150 mM NaCl, and
0.05% Tween 20)), washed, and resuspended as a 1:3 slurry in binding
buffer. Immunoprecipitation was carried out by mixing 30 µl of this
slurry with 25 µg of chromatin-associated protein extract and an
equal volume of IP buffer (25 mM Tris-Cl, pH 7.3, 125 mM NaCl, 1% Triton X-100 plus protease inhibitor mixture) overnight at 4 °C. Protein concentrations were determined using Bradford reagent (Bio-Rad). The protein A- or G-agarose bead-bound immune complexes were then washed three times with IP buffer and resuspended in this buffer. The IPs and aliquots of
chromatin-associated extracts from the cell lines examined were
subjected to SDS-PAGE, using 8-16% gradient gels or 10% gels. After
electrophoresis, the proteins were transferred to nitrocellulose and immunoblotted.
SpII*, anti-
spectrin, anti-FANCA (carboxyl-terminal), anti-FANCC (amino-terminal)
(generous gift of Dr. Alan D'Andrea, Harvard Medical School), and
anti-topoisomerase II (Oncogene). When blots were reprobed, they were
stripped of IgM using a high salt buffer (20 mM Tris, pH
7.5, 500 mM NaCl, and 0.3% Tween 20) and IgG per the
manufacturer's instructions (Pierce) and were reblocked before
reprobing with primary antibody. Alternatively, blots were incubated
with secondary antibody conjugated to alkaline phosphatase in 1%
nonfat dried milk blocker (Bio-Rad) in TTBS, washed in TTBS and
incubated in 5-bromo-4-chloro-3-indoyl phosphate/nitro blue tetrazolium
solution (Bio-Rad). Images were scanned using a Hewlett-Packard ScanJet
4c/T scanner and analyzed using ImageQuant (Molecular Dynamics).
RESULTS
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DISCUSSION
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spectrin ( spectrin II). The calculated peptide masses obtained from the protein digest were searched against two
different data bases to determine the number of matched peptides. A
ProFound search of the peptides obtained from the 230-kDa protein digest gave a probability score of 1.0e+00 to human spectrin II
with a clean break between this score and the score of the next
nonrelated protein. The percentage coverage of the known sequence for
this protein was 35% (i.e. the peptide masses obtained for
analysis matched to 35% of the known protein sequence). This exceeded
the minimum percentage coverage of 25% set as a criterion for a
protein match by the Keck Facility. A second ProFound search was
performed, after deleting sequences that matched human spectrin II,
and no additional protein was identified. PeptideSearch matched the
same protein with 33% coverage. This analysis of the 230-kDa protein
was repeated on a second sample obtained from another HeLa cell
chromatin-associated protein extract with the same result. Because the
isoform of the spectrin we have identified is unknown, this protein
has been designated SpII* in accordance with proposed nomenclature (17, 28).
SpII* was made by Western
blot analysis. A mAb against an spectrin, with specificity for
mammalian nonerythroid spectrin (Chemicon), bound to the 230-kDa
protein from HeLa chromatin-associated protein extracts (Fig.
1, lane 1); binding of this
mAb to human erythroid spectrin ( and 
chains) (Fig. 1,
lane 2) was used as a control. Similarly, anti-SpII*
bound to spectrin in HeLa cell extracts and to the and chains of erythroid spectrin (Fig. 1, lane 4). Both anti-
spectrin and anti-SpII* showed cross-reactivity with and spectrin. Using these antibodies, spectrin was not detected in the
HeLa cell chromatin-associated protein extracts. Confirmation that both
mAbs were binding to the same protein band was made by analysis of the
respective immunoblots lined up with gold-stained proteins from a
section of the same membrane (data not shown). Further confirmation of
the location of spectrin on these gels was obtained by Western blot
analysis using a mAb that principally recognized erythroid spectrin
(data not shown).
View larger version (39K):
[in a new window]
Fig. 1.
Confirmation of the identity of the 230-kDa
protein as nonerythroid spectrin.
Western blot analysis was carried out on the binding of anti-
spectrin (Chemicon) (1:8,000 dilution) (lanes 1 and
2) and anti-p230 (lanes 3 and 4) to
chromatin-associated proteins from HeLa cells (lane 1 and
3) or to erythroid spectrin (lanes 2 and
4). The blot was first probed with anti-p230 and then
stripped and reprobed with anti- spectrin.
SpII* is not one of the major bands present in these protein extracts (Fig. 2, A, lane 1 and
B). The SpII* band (Fig. 2A, lane
1) lined up exactly with the spectrin band from erythroid
spectrin (Fig. 2A, lane 2), which was run
alongside it on the same gel. The identity of these bands was verified
by Western blot analysis. Both proteins, according to electrophoretic
mobility, had an apparent molecular mass of approximately 230 kDa,
which agrees with previously reported values (230-240 kDa) (18, 29)
and is lower than the calculated molecular mass of 284 kDa for
nonerythroid and 280 kDa for erythroid spectrin (18, 30).
View larger version (79K):
[in a new window]
Fig. 2.
Electrophoretic separation of the HeLa cell
chromatin-associated protein extracts. The HeLa cell extracts were
separated by SDS-PAGE on a 15 × 20-cm gel and stained with SYPRO
orange. A, a 7-9.5% gradient gel for separation of
proteins above 100 kDa. Lane 1, HeLa cell extract;
lane 2, erythroid spectrin. B, a 12.5-15%
gradient gel for separation of proteins below 100 kDa.
Asterisks indicate the same bands on different gels.
Immunoblotting showed that there are reduced levels of the 230-kDa
protein in chromatin-associated protein extracts from FA complementation group B (FA-B), C (FA-C), and D (FA-D) cells (Fig. 3, top panel, lanes
3-5) compared with normal extracts (Fig. 3, top panel,
lane 1) just as there are in the FA-A extracts (Fig. 3,
top panel, lane 2). Thus in at least four FA
complementation groups there is a deficiency in SpII*. In these
studies topoisomerase II was used as an internal control (Fig. 3,
bottom panel).
|
Levels of SpII* are restored to normal in FA-A cells that have
been corrected with FANCA cDNA (Fig.
4, top panel, lanes 1-3), as previously shown (16), indicating that the
FANCA gene plays a role in the stability or expression of
this protein. Similar results were obtained when immunoblotting was
carried out using anti- spectrin (Chemicon). Reduced binding of
anti- spectrin to SpII* in FA-A extracts was restored to
normal in corrected FA-A cells (Fig. 4, second panel,
lanes 1-3). Topoisomerase II was used as an internal
control.
|
To determine whether SpII* interacts with the FANCA and FANCC
proteins, immunoprecipitation studies were carried out. Anti-FANCA immunoprecipitation and immunoblotting with either anti-SpII* or
anti- spectrin demonstrated that SpII* co-immunoprecipitated with FANCA and FANCC from HeLa chromatin-associated protein extracts (Fig. 5, lane 1). Preliminary
studies indicate that FANCG also immunoprecipitated with FANCA and
FANCC. None of these proteins co-immunoprecipitated from FA-A extracts,
which lack FANCA and are deficient in SpII* (Fig. 5, lane
2). In extracts from corrected FA-A cells, SpII* again
co-immunoprecipitated with FANCA and FANCC (Fig. 5, lane 3)
and also FANCG (preliminary data). These results thus show that
SpII* forms a nuclear complex with these proteins. Faint bands
were detected in the pre-immune precipitations (Fig. 5, lane
4) indicating a slight reactivity of the pre-immune sera with
spectrin.
|
The binding of FANCA and FANCC to SpII* was confirmed by anti-
spectrin immunoprecipitation (Fig.
6A, lane 1).
Anti- spectrin was used, even though it may be against a different
isoform than our SpII*, because our anti-SpII* is of the
IgM class and cannot be used effectively in immunoprecipitations. In
FA-A extracts, no FANCA and reduced amounts of SpII* and FANCC
immunoprecipitated (Fig. 6A, lane 2). This
corresponds to no expressed FANCA (Fig. 6B, lane
2) and reduced levels of SpII* (Fig. 4, top
panel, lane 2) and FANCC (Fig. 6B,
lane 2) in FA-A nuclei. Reduced levels of FANCC in FA-A cell
nuclei have also been reported by Yamashita et al. (36). In
extracts from corrected FA-A cells, SpII*, FANCA, and FANCC again
co-immunoprecipitated (Fig. 6A, lane 3). These
studies confirm that FANCA and FANCC form a complex with SpII* in
the nucleus and that normal levels of SpII* and FANCA in the
nucleus are important for complex formation.
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DISCUSSION |
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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SpII* has now been identified as associated with a protein
complex that we have isolated from normal human cells and shown to be
involved in repair of DNA interstrand cross-links (12-15). The exact
isoform of this protein is not yet know and is currently being
examined. Isoforms of each spectrin gene, generated by alternative pre-mRNA splicing, have been shown to occur (17, 18, 30). The
presence of different isoforms in different cells and even in the same
cell suggests distinct functions for each (18, 30, 31).
Anti-SpII* like anti- spectrin had cross-reactivity with spectrin. This probably indicates a common epitope that is being
recognized by these antibodies. This is not unusual, because genes for
nonerythroid and spectrin encode proteins that are
approximately 58-60% identical to their erythroid counterparts (18,
28, 30, 32), and between nonerythroid and erythroid and spectrin there is also significant homology (18, 33).
The reduced levels of SpII* that we observe in FA-A, FA-B, FA-C,
and FA-D cells correlate with the decreased levels of DNA repair
synthesis (i.e. unscheduled DNA synthesis) that are observed in these cells in response to DNA interstrand cross-linking agents and
with the reduced ability of the protein complex to incise cross-linked
DNA (13, 16). These values are approximately 25-35% of normal cells
(16). Because we have shown that this protein complex contains a number
of the proteins involved in nucleotide excision repair (i.e.
XPR, RPA, TFIIH, HHR23B, XPG, ERCC1, XPF, and PCNA) (16), it is
possible that a role for SpII* in the repair process is to act as
a scaffold to align these proteins so as to enhance their interactions.
This alignment could be particularly important in repair of interstrand
cross-links where recombination may be involved (34, 35). Reduced
levels of SpII* in the nucleus would thus be expected to reduce
the efficiency of the repair process rather than inhibit it altogether,
consistent with our experimental findings.
The FANCA and FANCC proteins have been shown to form a complex in the
nucleus (36, 37). The present results show that SpII* also forms
a complex in the nucleus with these proteins. Because studies suggest
that FANCA and FANCC may not bind directly to each other in the nucleus
but that their interaction may involve another, as yet unknown, protein
(36), it is possible that SpII* is this protein. These finding
suggest that SpII* may act as a scaffold to align or enhance
interactions between these two FA proteins, possibly other FA proteins
such as FANCG, and proteins involved in DNA repair as well as in other
cellular processes. In addition, because levels of SpII* are
reduced in cells from at least four FA complementation groups (FA-A,
FA-B, FA-C, and FA-D), this suggests that these FA genes may all be
involved in the stability or expression of this protein. This is
further demonstrated by the finding that in FA-A cells expressing the
FANCA cDNA, this deficiency in SpII* is corrected
as is the DNA repair defect (16).
The role of nonerythroid spectrin in the nucleus is not completely
understood. Nonerythroid -spectrin has been shown to be associated
with the nuclear matrix (38). A number of studies suggest that the
nuclear matrix is important in DNA repair (39-41). SpII* may
link the repair process to the nuclear matrix. spectrin has also
been shown to interact with a number of proteins (17, 18) and
potentially some of these interactions could occur in the nucleus. For
example, spectrin contains a calmodulin binding site and has been
shown to associate with this protein (19-21) and it also has a SH3
domain, which interacts with tyrosine kinases (17, 20, 22).
Collectively these proteins, which potentially could bind to
SpII* in the nucleus, have been shown to interact with proteins
involved in DNA synthesis, cell cycle progression, mitosis, gene
expression, and signal transduction (17, 19-22, 42). spectrin has
also been shown to be involved in cell growth and differentiation (17,
18). A deficiency in SpII* in FA cells could thus have far
reaching consequences due to the number of systems affected. This could
possibly explain some of the diverse cellular and clinical defects
reported in FA, such as cell cycle defects, aberrant induction of
apoptosis, and developmental abnormalities (1-3). Identification of
other FA genes and their products should help determine how many of the
FA proteins form a complex with SpII* and whether these proteins
also play a role in the stability or expression of SpII*.
Elucidation of this relationship may help delineate the function of
these FA proteins in the nucleus, the nature of their interaction with
SpII*, and their role in the deficiency in SpII* observed
in FA cells.
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ACKNOWLEDGEMENTS |
|---|
We thank Robert Lockwood for culturing of the human cell lines and Dr. W. Clark Lambert for critically reviewing the manuscript.
| |
FOOTNOTES |
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* This work was supported by National Institutes of Health Grants R01 HL54806 (to M. W. L.) and CA09665 (to L. W. M.) and by a Translational Leukemia of America Award (to C. E. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Pathology
and Laboratory Medicine, UMDNJ-New Jersey Medical School, 185 S. Orange
Ave., Newark, NJ 07103. Tel.: 973-972-4405; Fax: 973-972-7293; E-mail:
mlambert@umdnj.edu.
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ABBREVIATIONS |
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The abbreviations used are: FA, Fanconi anemia; mAb, monoclonal antibody; IP, immunoprecipitation; MALDI-MS, matrix-assisted laser desorption ionization mass spectrometry; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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