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Analyses of PRMT1 proteins in human colon tissues from
Hirschsprung disease patients
T.-T. WU,*,� T.-W. TSAI,� Y.-T. SHEN,� J.-D. HSU,§ L.-C. YANG§ & C. LI�
*Department of Pediatric Surgery, Chung Shan Medical University Hospital, Taichung, Taiwan
�School of Medicine, Chung Shan Medical University, Taichung, Taiwan
�Department of Biomedical Sciences, Chung Shan Medical University, Taichung, Taiwan
§Department of Pathology, Chung Shan Medical University Hospital, Taichung, Taiwan
Abstract
Background Protein arginine methyltransferase 1
(PRMT1) catalyzes the majority of arginine methyla-
tion in cells and plays important roles in the differ-
entiation and development of neurons. It is also
implicated in the regulation of nitric oxide synthetase
(NOS). Hirschsprung disease (HSCR) is characterized
by the absence of intramural ganglion cells in the
nerve plexuses of the distal gut. Methods Western blot
analyses revealed reduced PRMT1 protein levels in the
aganglionosis segments of HSCR patients. Immuno-
histochemistry detected PRMT1 expression in the
colonic mucosa, the enteric nervous system (ENS) and
endothelial cells. Specific and strong PRMT1 expres-
sion in neuron cell bodies of the plexus was demon-
strated by immunofluorescent double-labeling with
neuron-specific marker HuC/D. Key Results In the
mucosa, PRMT1 was detected at all crypt cells.
Intensive PRMT1 staining was detected in the sub-
mucosal and the myenteric plexuses in normal or
oligoganglionosis segments. Aganglionosis segments
from HSCR patients contain no plexuses, and thus not
labeled with PRMT1. The phenomenon is specific to
the megacolon of HSCR as strong PRMT1 staining was
observed in plexuses of the rectal ectasia segments
(dilated rectum and distal sigmoid not related with
aganglionosis) from anorectal malformation patients.
Furthermore, PRMT1 was also present in the same
neuronal cells expressing neuronal NOS in the plex-
uses. Conclusions & Inferences We suggest that
PRMT1 can be a useful marker for HSCR. This study is
the first illustration of PRMT1 protein expression in
human tissues from non-cancerous disease and set up
the base for further investigations of PRMT1 function
in ENS development and intestinal motility.
Keywords anorectal malformations, Hirschsprung
disease, nitric oxide synthetase, protein arginine
methyltransferase 1.
INTRODUCTION
Protein arginine methyltransferases (PRMTs) are
enzymes that catalyze the transfer of a methyl group
from S-adenosylmethionine (SAM) to guanidine nitro-
gens of specific arginines in proteins.1 Protein arginine
methyltransferase 1 is a predominant type I methyl-
transferase for arginine methylation to form monome-
thylarginine (MMA) and asymmetric dimethylarginine
(ADMA). Proteolytic degradation of arginine methylat-
ed proteins represents the major source of free MMA or
ADMA, which are endogenous inhibitors of nitric oxide
synthases (NOS).2 Besides a well-known endothelial-
derived relaxing factor, nitric oxide (NO) is also an
important neurotransmitter in intestine, mediating
nonadrenergic noncholonergic inhibitory transmis-
sion.3 Protein arginine methyltransferase 1 thus might
play a regulatory role for NO synthesis in intestine
through the formation of ADMA containing proteins
and later free ADMA through protein degradation.
In mammals, PRMT1 has a wide substrate spectrum
including histones, heterogeneous nuclear ribonucleo-
proteins (hnRNPs) and various RNA binding proteins.
Protein arginine methyltransferase 1 and the protein
methylation it catalyzes are involved in various impor-
tant biological processes such as signal transduction,
Address for correspondence
Dr. Chuan Li, Department of Biomedical Sciences, ChungShan Medical University, 110 Sec. 1, Chein-Kuo North Road,Taichung, Taiwan.Tel: 886 4 2473 0022 ext 11807; fax: 886 4 2475 7412;e-mail: cli@csmu.edu.twReceived: 11 August 2009Accepted for publication: 18 April 2010
Neurogastroenterol Motil (2010) 22, 984–e254 doi: 10.1111/j.1365-2982.2010.01523.x
� 2010 Blackwell Publishing Ltd984
RNA processing, transcriptional regulation and even
DNA repair.4 It is also important for early embryogen-
esis,5 and neuronal differentiation.6
Even though ubiquitous expression of PRMT1 RNA
or protein was illustrated in various animal models
including rat,7 mouse,8 and fish,9 direct demonstration
of PRMT1 expression in human tissues are few and are
restricted to tumor samples.10,11 In this study colon
tissue samples from Hirschsprung disease (HSCR)
patients are examined for the expression of PRMT1.
Hirschsprung disease or aganglionic megacolon, the
most common congenital intestinal obstruction, is a
developmental disorder of the enteric nervous system
(ENS) characterized by the absence of intramural
ganglion cells in the nerve plexuses of the distal
gut.12 The ENS derived from migratory neural crest is
the largest subdivision of the peripheral nervous sys-
tem to regulate the gut behaviors, including the
coordination of the smooth muscle contractions of
the gut wall. The ENS neurons and glia cells organized
into ganglia, which are interconnected to form two
types of plexuses: the submucosal (Meissner’s) and the
myenteric (Auerbach’s) plexus between the longitudi-
nal and circular smooth muscle layers. Genes encoding
proteins of the RET signaling pathway (RET, GDNF
and NTN),13–17 participating in the endothelin type B
receptor pathway (EDNRB, EDN3, and ECE-1),18–20
involving transcriptional regulations such as the SRY-
box 10 gene (SOX10),21 and the zinc finger homeobox
1B22 have been reported to be related to the etiology of
HSCR. The importance of signaling pathways guiding
the migration of ENS cells to the terminal region of the
gut can be best exemplified by this disease.
Anorectal malformations (ARM) often associate
with rectal ectasia, a state of massive dilation of the
rectum and distal sigmoid not related with aganglion-
osis as in HSCR. We compared PRMT1 protein
expression in normal and dilated colon segments from
HSCR and ARM patients with rectal ectasia. This
study is the first illustration of PRMT1 protein
expression in human tissues from non-cancerous
disease and initiates the investigation of putative
PRMT1 function in ENS development and intestinal
motility.
MATERIALS AND METHODS
Human tissues
Tissues samples were obtained from eight newborns diagnosed atinfancy (five HSCR and three ARM associated with rectalectasia). Samples from HSCR patients were collected fromaganglionated, oligoganglionated and normal ganglionated
segments. Samples from ARM patients associated with rectalectasia were collected from abnormal distal, dilated and normalsegments. Hirschsprung disease patients received definite pull-through operation at Chung-Shan Medical University Hospitaland ARM patients associated with rectal ectasia were includedin our previous genetic analyses.23,24 The study was approved bythe local Institutional Research Board. Tissue samples werecollected immediately after operation and were stored at )80 �Cuntil use.
Western blot
Colon tissues were weighed, washed two times with phosphatebuffered saline (PBS), and then cut into small slices, resuspendedinto three times (vol/wt) lysis buffer [5% glycerol, 1 mM EGTA,1 mM DTT, 0.5% Triton X-100 and Complete EDTA-freeprotease inhibitor cocktail (Roche, Mannheim, Germany) inPBS] and homogenized with a homogenizer (T10 basic, IKA). Theprotein amount in the extracts was quantified by bicinchoninicacid assay (BCA) (Thermo Scientific, Waltham, MA, USA). Theextracted total proteins (30 lg) was separated by SDS polyacryl-amide gel electrophoresis and transferred to the nylon mem-brane. The membrane was then incubated with primaryantibodies (anti-PRMT1 antibodies; Upstate, 1 : 1000 dilution/anti-b-actin antibodies; Santa Cruz Biotechnology Inc, SantaCruz, CA, USA; 1 : 5000 dilution) at room temperature for60 min. For detection, HRP-conjugated secondary antibody (goatanti-rabbit/mouse-HRP, Santa Cruz Biotechnology Inc) andImmobilon Western (Millipore, Billerica, MA, USA) or VisGlow(Visual Protein Biotechnology Corp, Taiwan) chemiluminescentreagent were used.
Immunohistochemistry
Bowel tissue sections of full thickness and normal segments werefrom typical rectosigmoid HSCR patients and ARM patients withrectal ectasia. Immunohistochemical studies were performed onparaffin-embedded tissue cross sections. The slides were baked at50 �C overnight, soaked in xylene for 10 min three times, 100%ethanol for 5 min, 95% ethanol for 5 min, then 80% and 70%ethanol for one minute each. After rehydration for 5 min, theslides were boiled in citrate buffer (pH 6.1, Target RetrievalSolution; Dako, Carpinteria, CA, USA) for 25 min, washed withwater and PBS, treated with 1–3% H2O2 for 10 min, then washedagain with PBS. The slides were then incubated with the anti-PRMT1 antibodies (Upstate, 1 : 100 dilution) at room temperaturefor 30 min. After PBS wash for 5 min two times, the slides wereincubated with HRP-conjugated secondary antibody (goat anti-rabbit-HRP, Santa Cruz Biotechnology Inc) at room temperaturefor 30 min. Color was developed at room temperature with theSubstrate-Chromogen Solution (Dako LSAB2 System) for 5 minand then counter stained with Mayer hematoxylin, (MERCK,Darmstadt, Germany) for 1 min. The slides were dehydrated in50%, 75% and 100% ethanol for 1 min each, and sealed withCytoseal 60 Mounting medium (Thermo Scientific).
For immunofluorescence, de-paraffined slides were boiled incitrate buffer (pH 6.1, Target Retrieval Solution, Dako) for 25 min.The slides were then incubated with anti-PRMT1 antibodies(Upstate, 1 : 250 dilution) and anti-HuC/D (Molecular Probes,Carlsbad, CA, USA; 1 : 40 dilution) or anti-nNOS (BD Transduc-tion Laboratories, Franklin Lakes, NJ, USA; 1 : 250 dilution)antibodies at 4 �C for 12–16 h. After PBS washing, slides wereincubated with Rhodamine-conjugated anti-mouse IgG (JacksonImmunoResearch, West Grove, PA, USA; 1 : 200 dilution) andFITC-conjugated anti-rabbit IgG (Jackson ImmunoResearch, West
Volume 22, Number 9, September 2010 Analyses of PRMT1 proteins in human colon tissues
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Grove, PA, USA; 1 : 200 dilution) at room temperature for 2 h.Cells were rinsed again, incubated with DAPI (0.5 mg mL)1;Roche) at room temperature for 1 min and mounted in ibidireagent (ibidi GmbH, Germany).
RESULTS
PRMT1 expression in HSCR and ARM bywestern blot analyses
The expression levels of PRMT1 in the colon tissues
were evaluated first by western blot analyses. As
shown in Fig. 1, the amount of PRMT1 protein
expressed in the normal ganglionic and oligoganglionic
colon specimens from HSCR patients were compara-
ble, while that expressed in aganglionic segments was
reduced. Detection of the same blot with anti-b-actin
antibodies confirmed equal protein loading. Fig. 1A
represents the western blot result of colon tissue
samples from a typical short-segment agangliosis
HSCR patient. Similar results were obtained from
samples of other HSCR patients.
Interestingly, similar amount of PRMT1 protein was
expressed in normal, dilated and abnormal distal
segments of ARM patients (Fig. 1B). The results indi-
cate that the reduced PRMT1 expression in the abnor-
mal bowel segments might be specific to HSCR.
Immunohistochemical detection of PRMT1 inHSCR
The histological appearance of the colon specimens
from HSCR patients and the expression of PRMT1 in
the normal ganglionic, oligoganglionic (dilated) and
aganglionic (distal) segments are shown in Fig. 2.
Protein arginine methyltransferase 1 was detected in
mucosa, submucosa and the inner circular and outer
longitudinal smooth muscle layers in sections from
normal, oligoganglionic and even aganglionic segments
of HSCR patients (Fig. 2A, B, C).
In the mucosa, strong PRMT1 staining was observed
in the crypts in all three segments (Fig. 2D, E, F). In the
submucosa, the expression of PRMT1 in human cap-
illary endothelial cells (arrowheads in Fig. 2G, H, I) was
clearly detected. Intensive PRMT1 staining was de-
tected in submucosal plexuses in the normal gangli-
onic and oligoganglionic but not aganglionic segments
(arrows in Fig. 2G, H). Smooth muscle fibers from the
longitudinal and circular muscularis were barely
stained in the cytoplasm but clearly stained in the
nuclei (Fig. 2J, K, L). The most observable difference
between normal and aganglionic tissues is that PRMT1
was significantly detected in the submucosal (Meiss-
ner’s) and the myenteric (Auerbach’s) plexuses only in
normal or oligoganglionic segments (Fig. 2J, K). In
aganglionic segments, the plexuses were not formed
and not labeled with PRMT1 (Fig. 2L). This result
probably can explain the reduced level of PRMT1
detected by western blot in the agangliosis samples.
Immunohistochemical detection of PRMT1in ARM
The tissue samples from ARM patients associated with
rectal ectasia were divided into three segments: nor-
mal, dilated, and distal abnormal. As shown in Fig. 3,
PRMT1 expression patterns in ARM normal (Fig. 3A,
D, G, J), dilated (Fig. 3B, E, H, K) and abnormal distal
(Fig. 3C, F, I, L) segments were similar to those of
normal ganglionic segments from the HSCR patients.
Immunofluorescent analysis of PRMT1expression in ganglions
As PRMT1 stained clearly in the ganglions in the
normal colon segments from HSCR patients, we
further compared its expression pattern with HuC/D
by immunofluorescent double labeling (Fig. 4). HuC/D
is a neuron-specific RNA binding protein and serves as
a specific neuron marker. Protein arginine methyl-
transferase 1 staining fully overlapped with HuC/D
staining in the plexus (Fig. 4C). DAPI is a fluorescent
stain that can bind strongly with DNA and then stains
clearly the nuclei of all cells. Merge of the PRMT1 and
DAPI signals showed that PRMT1 was absent in some
cells that also can not be stained by Hu in the plexus
(Fig. 4D, indicated by arrows; the same region was also
indicated in Fig. 4A, B).
N
Normal
PRMT1
Actin
PRMT1
Actin
O
Dilated
A
Abnormal
A
B
Figure 1 Protein arginine methyltransferase 1 (PRMT1) expression in
Hirschsprung disease (HSCR) and anorectal malformation (ARM) by
western blot analyses. (A) Protein extracts from normal ganglionic,
oligoganglionic and aganglionic (represented as N, O and A) colon
specimens in HSCR were probed by western blot analysis with PRMT1
antibodies. (B) Protein extracts from normal, dilated and abnormal
colon specimens in ARM were probed by western blot analysis with
PRMT1 antibodies. Detection of with an anti-b-actin antibody con-
firmed equal protein loading.
T.-T. Wu et al. Neurogastroenterology and Motility
� 2010 Blackwell Publishing Ltd986
We then stained the ganglion with neuronal NO
synthase (nNOS) (Fig. 5A). The nNOS and DAPI
staining clearly demonstrated the cytosolic expression
of nNOS in the neurons in the ganglions (Fig. 5C).
Protein arginine methyltransferase 1 appeared to be
present in the same cells expressing nNOS with
cytoplasmic as well as nuclear expression (Fig. 5B, D).
The results further confirm the specific expression of
PRMT1 in the neuron cells. Strong detection of
PRMT1 in the nuclei of neurons and neighboring
muscle fibers also clearly demonstrated its concen-
trated nuclear expression and dilute cytoplasmic stain-
ing. Similar results were obtained in the plexuses from
normal (data not shown) and dilated segments from
ARM patients (Figures S1 and S2).
DISCUSSION
In this study we observed PRMT1 expression in
different cell types of human colon tissues from HSCR
and ARM patients. We showed clear and significant
PRMT1 staining in the submucosal and myenteric
plexuses of normal colon tissues, the first direct
demonstration of PRMT1 expression in human peri-
pheral nervous system. The results are consistent with
PRMT1 functions in neural development and differen-
tiation. In the gut, NO is synthesized mainly by the
nNOS in the submucosal and myenteric plexus.25
Significant expression of PRMT1 in these regions is in
agreement with the possibility that PRMT1 can serve
as a regulator for NOS in intestine through the
A B C
D E F
G H I
J K L
Figure 2 Immunohistochemical detection of protein arginine methyltransferase 1 (PRMT1) in Hirschsprung disease. Overall view of the intestinal
wall in (A) normal ganglionic, (B) oligoganglionic, and (C) aganglonic colon specimens were illustrated with PRMT1staining. Mucosal, submucosal
and muscularis staining in normal ganglionic (D,G,J), oligoganglionic (E,H,K) and aganglonic (F,I,L) colon specimens are shown. Positive stainings in
all crypts, capillary endothelial cells (arrowhead), submucosal and myenteric plexuses (arrows) are indicated. Absence of staining of hypertrophic
nerve bundles is indicated by asterisk. Scale bars: A–C: 500 lm; D–L: 50 lm.
Volume 22, Number 9, September 2010 Analyses of PRMT1 proteins in human colon tissues
� 2010 Blackwell Publishing Ltd 987
formation of ADMA. Moreover, immunofluorescent
double labeling analysis confirmed concentrated
expression of PRMT1 specifically in the cell bodies of
neurons in the plexus. Co-localization of PRMT1 and
nNOS in the same neuron cells also strengthens their
putative functional correlation.
Furthermore, the absence of submucosal and myen-
teric plexuses in the aganglionic segments from HSCR
patients can be revealed by PRMT1 staining. Identifi-
cation of the ganglions is critical for the diagnosis of
HSCR. Examination of HE-stained sections, acetylcho-
linesterase histochemical analysis or immunochemi-
cal detection with marker genes such as S100 (glia
marker), NeuN (neuron marker) or RET have been
conducted.26–28 As PRMT1 stained most strongly in
both the crypts and plexuses, PRMT1 can serve as a
marker for HSCR. Most of the HSCR markers are
neuron-specific. With these markers, negative staining
of HSCR sections due to aganglionosis can be con-
cluded only after the exclusion of unsuccessful immu-
nohistochemical detection. On the contrary, PRMT1
signals in the mucosa crypts are intense in both normal
and aganglionic segments and can serve as an internal
positive control. Furthermore, we showed that the
effect is specific to the megacolon due to HSCR but not
other bowel disorders with dilated rectum such as
ARM with rectal ectasia. Specifically, both submucosal
and myenteric plexuses can be readily detected by
A B C
D E F
G H I
J K L
Figure 3 Immunohistochemical detection of protein arginine methyltransferase 1 (PRMT1) in anorectal malformation. Overall view of the intestinal
wall in (A) normal, (B) dilated, and (C) abnormal colon specimens were illustrated with PRMT1staining. Mucosal, submucosal and muscularis
staining in normal (D,G,J), dilated (E,H,K) and abnormal distal (F,I,L) colon specimens are shown. Positive staining in all crypts, capillary endothelial
cells (arrowhead), submucosal and myenteric plexuses (arrows) are indicated. Scale bars: A–C: 500 lm; D–L: 50 lm.
T.-T. Wu et al. Neurogastroenterology and Motility
� 2010 Blackwell Publishing Ltd988
PRMT1 in normal and dilated colon tissues from ARM
patients with rectal ectasia.
The expression pattern of PRMT1 in human colon
tissues from HSCR and ARM can be compared with
the PRMT1 expression in colon cancer samples.11
Even though Mathioudaki et al. reported that PRMT1
proteins reside mainly in cytoplasm and rarely in
nucleus in both normal and colon cancer tissues by
immunohistochemistry, we observed concentrated
PRMT1 staining in the nuclei of cells in all layers
of the bowel. Significant cytoplasmic PRMT1 staining
was observed in cells in the crypts and plexuses. On
the other hand, dilute staining in the cytoplasm of
muscle cells was observed with higher magnification.
Immunofluorescent double labeling experiments with
DAPI or nNOS also clearly demonstrated the concen-
trated nuclear expression of PRMT1 in neurons.
Controversial nuclear or cytoplasmic localizations of
PRMT1 have been reported. Protein arginine methyl-
transferase 1 can shuttle between nucleus and cyto-
plasm and inhibition of methylation results in nuclear
accumulation.29 Recently PRMT1 subcellular locali-
zation appeared to be related to the isoforms of
PRMT1,30 the cell-type,31 and might be affected with
interacting proteins.32 It can not be excluded that
different PRMT1 antibodies might recognize different
isoforms of the proteins and thus led to the contra-
dictory results. Nevertheless, our results provide more
detailed information of the localization of PRMT1
protein in different bowel cell types, especially in the
neurons but not other cells of the ganglions by the
immunofluorescent analyses.
A few genes involved in signaling pathways of the
receptor tyrosine kinase RET or endothelin type B
receptor and in transcriptional regulations for ENS
development have been reported to be involved in the
A B
C D
Figure 4 Immunodetection of HuC/D and
protein arginine methyltransferase 1
(PRMT1) in myenteric plexuses in normal
colon sections from an Hirschsprung disease
patient. (A–D) Double labelling with HuC/
D, PRMT1 and DAPI. (A) HuC/D: staining
of ganglion cell bodies, (B) PRMT1: staining
of ganglion cell bodies and nuclei of muscle
cells, (C) HuC/D/PRMT1 Merge, (D)
PRMT1/DAPI Merge: absence of staining in
the non-ganglion cells (arrows). Scale bar:
50 lm.
A B
C DFigure 5 Immunodetection of neuronal NO
synthase (nNOS) and protein arginine
methyltransferase 1 (PRMT1) in myenteric
plexuses in normal colon sections from an
Hirschsprung disease patient. (A–D) Double
labelling with nNOS, PRMT1 and DAPI. (A)
nNOS: staining of ganglion cell bodies, (B)
PRMT1: staining of ganglion cell bodies and
nuclei of muscle cells, (C) nNOS/DAPI
Merge, (D) nNOS/PRMT1 Merge. Scale bar:
50 lm.
Volume 22, Number 9, September 2010 Analyses of PRMT1 proteins in human colon tissues
� 2010 Blackwell Publishing Ltd 989
etiology of HSCR.12 Nevertheless, mutations in these
genes can only explain a small portion of the HSCR
patients. Genes involved in epigenetic regulations
including DNA methylation and histone modifications
can act as genetic modifiers. As PRMT1 is responsible
for the methylation of histone H4 arginine 3 methyl-
ation,4 and plays critical roles in the differentiation and
development of neuron cells,5,6 protein arginine meth-
yltransferase 1 might be related to the development of
ENS and be a potential modifier of HSCR. Even though
no different subcellular localization or expression
could be observed for PRMT1 in ganglions of HSCR
or ARM patients, illustration of PRMT1 expression
pattern in colon segments can be the basis for further
investigations of putative PRMT1 function in ENS
development and intestinal motility.
ACKNOWLEDGMENT
This study was supported in part by National Science Council(NSC 96-2314-B-040-024), Chung Shan Medical University(CSMU 96-OM-A-020) and Chung Shan Medical UniversityHospital (CSH-2009-C-008) to TTW, and National Science Coun-cil (NSC 93-3112-B-040-001 and 94-2320-B-040-044) to CL. Wethank the patients involved in this study.
AUTHOR CONTRIBUTIONS
TW collected the patients and designed the research andanalyzed the data; TT conducted the western blot and immu-nohistochemical analyses; YS performed immunofluorescentexperiments and helped preparing the manuscript; JH and LYhelped preparing tissue sample sections and immunohistochem-istry; CL designed the research, analyzed the data and wrote thepaper. Competing Interests: the authors have no competinginterests.
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SUPPORTING INFORMATION
Additional Supporting Information may be found in the online version of this article:
Figure S1. Immunodetection of HuC/D and protein arginine methyltransferase 1 (PRMT1) in myenteric plexuses
in dilated colon sections from an anorectal malformation patient. (A–D) Double labelling with HuC/D, PRMT1 and
DAPI. (A) HuC/D: staining of ganglion cell bodies, (B) PRMT1: staining of ganglion cell bodies and nuclei of muscle
cells, (C) HuC/D/PRMT1 Merge, (D) PRMT1/DAPI Merge. Scale bar: 50 lm.
Figure S2. Immunodetection of neuronal NO synthase (nNOS) and protein arginine methyltransferase 1 (PRMT1)
in myenteric plexuses in dilated colon sections from an anorectal malformation patient. (A–D) Double labelling with
nNOS, PRMT1 and DAPI. (A) nNOS: staining of ganglion cell bodies, (B) PRMT1: staining of ganglion cell bodies and
nuclei of muscle cells, (C) nNOS/DAPI Merge, (D) nNOS/PRMT1 Merge. Scale bar: 50 lm.
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Volume 22, Number 9, September 2010 Analyses of PRMT1 proteins in human colon tissues
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