Restricted localization of proline-rich membrane anchor
(PRiMA) of globular form acetylcholinesterase at the
neuromuscular junctions – contribution and expression
from motor neurons
K. Wing Leung, Heidi Q. Xie, Vicky P. Chen, Mokka K. W. Mok, Glanice K. Y. Chu, Roy C. Y. Choi
and Karl W. K. Tsim
Department of Biology and Center for Chinese Medicine, The Hong Kong University of Science and Technology, China
Acetylcholinesterase (AChE; EC 3.1.1.7) plays a cru-
cial role in terminating the synaptic transmission by
hydrolyzing the neurotransmitter acetylcholine at the
neuron-to-neuron synapses in the central nervous sys-
tem and at the neuromuscular junctions (NMJs) in the
peripheral nervous system. AChE exists in different
molecular forms. The formation of these molecular
forms depends on alternative splicing in the 3¢ region
of the primary transcript [1], which generates the
AChE
R
(‘readthrough’), AChE
H
(‘hydrophobic’) and
AChE
T
(‘tailed’) subunits, containing the same cata-
lytic domain but different carboxyl termini [1]. In
mammals, the AChE
R
variant produces a soluble
monomer that is up-regulated in the brain during
stress [2]; the AChE
H

Keywords
acetylcholinesterase; molecular form;
muscle fiber type; neuromuscular junction;
proline-rich membrane anchor
Correspondence
K. W. K. Tsim, Department of Biology,
The Hong Kong University of Science and
Technology, Clear Water Bay Road,
Kowloon, Hong Kong SAR, China
Fax: +852 2358 1559
Tel: +852 2358 7332
E-mail:
(Received 21 November 2008, revised 11
March 2009, accepted 25 March 2009)
doi:10.1111/j.1742-4658.2009.07022.x
The expression and localization of the proline-rich membrane anchor
(PRiMA), an anchoring protein of tetrameric globular form acetylcholines-
terase (G
4
AChE), were studied at vertebrate neuromuscular junctions.
Both muscle and motor neuron contributed to this synaptic expression
pattern. During the development of rat muscles, the expression of PRiMA
and AChE
T
and the enzymatic activity increased dramatically; however,
the proportion of G
4
AChE decreased. G
4
AChE in muscle was recognized

G
4
AChE) in brain and muscle [3–5]. Two PRiMA
isoforms (PRiMA I and PRiMA II) are generated
from the PRiMA gene by alternative splicing. PRi-
MA I contains a longer C-terminal cytoplasmic
domain than does PRiMA II [6].
Although asymmetric AChE is the predominant
species at NMJs and its appearance in muscle coin-
cides with the establishment of neuromuscular contacts
during development and regeneration [7,8], G
4
AChE
also exists in muscles. Several studies have revealed
that the level of G
4
AChE is controlled by the
dynamic activity of skeletal muscles. The transcrip-
tional regulation of PRiMA is down-regulated during
myogenic differentiation and under the influence of
innervation [9]. In line with the transcriptional expres-
sion of PRiMA, the proportion of G
4
AChE decreases
during myogenic differentiation and innervation [1,9].
In mammals, fast-twitch muscles contain a large
amount of G
4
AChE, whereas slow-twitch muscles
contain a much smaller amount [10].

NMJs.
Results
Regulation of G
4
AChE and PRiMA during muscle
development
A rabbit polyclonal antibody against the C-terminus
of PRiMA I was generated. To validate the PRiMA
antibody, a full-length mouse PRiMA cDNA (corre-
sponding to PRiMA I unless specified) and a C-termi-
nal truncated mutant (PRiMA
DC-term
) cDNA, both
tagged with a FLAG epitope, were transfected into
HEK293T cells. In western blot analysis, a FLAG
antibody recognized both PRiMA and PRiMA
DC-term
with protein bands of approximately 20 and 16 kDa,
respectively: these protein bands corresponded to the
predicted size of the recombinant proteins (Fig. 1A).
The PRiMA antibody, however, recognized only
the full-length PRiMA, but not the truncated
PRiMA
DC-term
construct. In addition, the recognition
was fully blocked by pre-incubation of the PRiMA
Fig. 1. The specificity of the PRiMA antibody. (A) Protein samples (40 lg) of HEK293T cells expressing FLAG-PRiMA or FLAG-PRiMA
DC-term
were analyzed by 12% SDS–PAGE. Both PRiMA and FLAG antibodies (Ab) were used to label the PRiMA proteins. In the blocking experiment,
excess amounts of recombinant PRiMA antigen (Ag) (from residues 114 to 153) at 5 lgÆmL

intracellular motif. These two PRiMA isoforms may
be distinguished by RT-PCR using primers flanking
exons 4 and 5. In rat muscles, PRiMA I was found to
be present, whereas PRiMA II was barely detectable
(Fig. 2B). For precise quantification, we used real-time
PCR with the same set of primers. In agreement with
the absence of PRiMA II in muscle, all the amplified
products revealed by real-time PCR corresponded to
PRiMA I. The mRNA level of PRiMA I was up-regu-
lated gradually in the early postnatal stages and dra-
matically in the adult stage (Fig. 2B). Meanwhile, the
level of AChE
T
mRNA increased gradually from the
early postnatal stage to the adult. Using PRiMA anti-
body, the PRiMA protein was detected in the muscles
of embryonic rats; its level increased after postnatal
day 10 to the adult (Fig. 2C). As reported previously,
Fig. 2. Developmental profiles of PRiMA, AChE
T
and G
4
AChE in skeletal muscles. (A) Splice variants of PRiMA mRNAs (PRiMA I and II) are
illustrated. PRiMA II contains an additional exon 4b. Arrows show the location of primers used for qualitative and real-time PCR analyses. (B)
Total RNAs were extracted from rat leg muscles at different developmental stages to perform RT-PCR for PRiMA I (145 bp) PRiMA II
(302 bp) and AChE
T
(671 bp). Adult rat brain served as a positive control. One representative result is shown (top). The bottom panel shows
the results of real-time PCR analysis of the mRNA expression of PRiMA I and AChE
T

4
forms was reduced and the asymmetric form of
AChE (A
12
) was increased (Fig. 2D). In order to quan-
tify the relative amount of PRiMA-linked G
4
AChE in
developing muscle, protein extracts at different develop-
mental stages were analyzed by sedimentation in sucrose
density gradients. The proportion of G
4
AChE was
determined from the peak area, relative to the area of
the entire sedimentation profile, and its activity was
given by the product of this proportion with the total
AChE activity. The amount of G
4
AChE in muscle
increased twofold from birth to adult (Fig. 2E).
PRiMA-linked G
4
AChE therefore increased during
muscle development.
Expression of PRiMA-linked G
4
AChE in
fast-twitch and slow-twitch muscles
In order to investigate the expression level of PRiMA
and PRiMA-linked G

enzyme was used as a control.
We analyzed the localization of PRiMA in sections
of tibialis and soleus muscle by immunohistofluores-
cence. NMJs were visualized by labeling the post-
synaptic acetylcholine receptor (AChR) with
a-bungarotoxin (shown in red or pseudo-blue) and the
presynaptic nerve terminal with synaptotagmin (SV48;
shown in red) in both types of muscle (Fig. 4). PRiMA
(shown in green) was expressed at the NMJs, and its
distribution was wider than that of AChE and AChR,
extending into a peri-junctional zone where neither
Fig. 3. Expression of PRiMA and G
4
AChE in different muscles. (A)
Samples of extracts from adult rat soleus and tibialis containing
40 lg of protein were loaded per lane for western blotting of PRiMA
protein. Adult rat brain served as a positive control. The bottom panel
shows the quantification of PRiMA protein. The results are
expressed as the ratio to soleus (basal) equal to unity; means ± stan-
dard error of the mean, n = 4. (B) The relative amount of PRiMA-
linked G
4
AChE was quantified by ELISA. Tissue lysates from rat
brain, tibialis and soleus containing equal AChE activities were loaded
onto an ELISA plate precoated with serial dilutions of PRiMA anti-
body for 2 h. The retained AChE activity was determined. (C) For
immunodepletion, 1 mL samples of extracts from adult rat brain, tibi-
alis and soleus were incubated with PRiMA antibody (10 lgÆmL
)1
)

T
proteins were determined. GAPDH served as a loading control. (C) Quantification of proteins (from B) and
AChE activity during development. (D) One milliliter samples of extract from adult rat spinal cord, with and without depletion by the PRiMA
antibody (as in Fig. 3C), were analyzed by sucrose density gradients. AChE activity was plotted as a function of the S value, estimated from
the position of the sedimentation markers. Enzymatic activities are expressed in arbitrary units, and representative sedimentation profiles
are shown. (E) G
4
AChE specific activity in the spinal cord at different developmental stages was quantified as in Fig. 2E. The results are
expressed as the ratio to the value obtained at P1 (basal) equal to unity; means ± standard error of the mean (SEM), n =4.
K. W. Leung et al. Proline-rich membrane anchor at neuromuscular junctions
FEBS Journal 276 (2009) 3031–3042 ª 2009 The Authors Journal compilation ª 2009 FEBS 3035
AChE nor AChR was present in either muscle fiber
type. However, the precise localization of PRiMA has
yet to be determined.
Presence of PRiMA-linked G
4
AChE in motor
neurons
At NMJs, AChE may originate from the muscle fiber
and ⁄ or from the motor neuron. In order to examine the
presence of PRiMA and PRiMA-linked G
4
AChE, rat
spinal cords were collected at early postnatal and adult
stages. Qualitative PCR indicated that both PRiMA I
and II transcripts were expressed in the spinal cord: the
PRiMA I transcript decreased slightly after birth, but
increased dramatically thereafter and was the predomi-
nant form in the adult, the PRiMA II transcript first
increased but disappeared in the adult (Fig. 5A). As a

AChE-positive cells in the ventral horn (Fig. 6B).
These PRiMA-stained cells were motor neurons, as
shown by their reactivity with an anti-choline acetyl-
transferase (anti-ChAT) antibody. This identification
was further supported by double staining of neuronal
nuclei with a neuronal marker (NeuN). In contrast,
no PRiMA was found in glial cells that were labeled
specifically with an antibody against glial fibrillary
acidic protein (GFAP) (Fig. 6B). These results clearly
show that PRiMA is synthesized by motor neurons in
the spinal cord.
Although motor neurons are able to synthesize
PRiMA and produce G
4
AChE, the restricted localiza-
tion of PRiMA-linked G
4
AChE at NMJs could still
be derived from three sources: muscle, Schwann cells
and ⁄ or motor neurons. In order to determine the local-
ization of PRiMA-linked G
4
AChE, sections of tibialis
muscle were triple stained for PRiMA, SV48 and
AChR. The staining of PRiMA was coincident with
that of SV48, rather than with that of AChR (Fig. 7,
left panel). Similar results were obtained with another
Fig. 6. Motor neurons in the spinal cord express PRiMA. (A) Sche-
matic diagram showing the lumbar region of the spinal cord (left).
The dorsal horn and ventral horn are indicated. The right panel

sciatic nerve was surgically removed. After 7 days, we
examined the expression of PRiMA in both spinal
cord (lumbar region) and tibialis muscles by real-time
PCR analysis: PRiMA mRNA (PRiMA I) was not
modified significantly in the tibialis, but was reduced
by over 60% in the spinal cord (Fig. 8A). In contrast,
the mRNA level of AChE
T
was decreased in both the
spinal cord and tibialis when compared with that of
the sham-operated control (Fig. 8A). At the protein
level, western blot analyses showed that PRiMA and
AChE
T
were reduced by about 50% after denervation
in both tissues (Fig. 8B). This is consistent with a
decrease in AChE enzymatic activity of about 50% in
the spinal cord and tibialis muscle (Fig. 8B). Sucrose
density gradient analyses showed a significant reduc-
tion of G
1
and G
4
forms in the spinal cord and of G
1
,
G
4
and A
12

largely immunoprecipitated with a PRiMA antibody.
However, a fraction of G
4
AChE was not
immunodepleted (Fig. 3C), even when the amount of
antibody was increased or with a second round of
immunodepletion (not shown). The interaction of this
fraction with the antibodies may be prevented by the
presence of partner(s) associated with the C-terminal
region of PRiMA. In addition, no G
4
AChE was
found in muscles of PRiMA knockout mice, implying
that all G
4
AChE in muscle is linked with the mem-
brane-anchoring protein PRiMA. During muscle devel-
opment, the amount of PRiMA-linked G
4
AChE
progressively increased from birth to the adult stage.
In addition, the expression of PRiMA and G
4
AChE
Fig. 7. Presynaptic localization of PRiMA at NMJs. Adult rat tibialis sections were triple stained with Alexa 647-conjugated a-bungarotoxin
(pseudo-blue), anti-synaptotagmin (SV48; red) or anti-SNAP-25 (red) antibodies, and anti-PRiMA (green), and examined by confocal micros-
copy. Merged images allow a comparison of PRiMA with presynaptic markers (PRiMA + SV48 ⁄ SNAP-25) and a postsynaptic marker (PRi-
MA + AChR). The distribution of PRiMA overlapped with that of SV48 and SNAP-25. Representative images are shown, n = 4. Bar, 20 lm.
K. W. Leung et al. Proline-rich membrane anchor at neuromuscular junctions
FEBS Journal 276 (2009) 3031–3042 ª 2009 The Authors Journal compilation ª 2009 FEBS 3037

sis. The mRNA levels of denervated muscles (Den) corresponding to PRiMA (top) and AChE
T
(bottom) were determined by PCR and normal-
ized to those of control (sham-operated) muscles. (B) Samples of extracts from control and denervated muscles containing 50 lg of protein
were loaded per lane for the western blotting of PRiMA and AChE
T
. GAPDH served as a loading control. The bottom panel shows the ratios
of AChE enzymatic activity after nerve section to control values. The results are expressed as the ratio to control values (sham-operated)
equal to unity; means ± standard error of the mean (SEM), n = 3. (C) Effect of nerve section on AChE molecular forms in the spinal cord
and tibialis muscles. Samples containing equal amounts of protein were loaded onto sucrose gradients. AChE activity was plotted as a func-
tion of the S value, estimated from the position of the sedimentation markers. Enzymatic activities are expressed in arbitrary units, and
representative sedimentation profiles are shown. (D) Sections from adult rat tibialis after 7 days of denervation (right) and sham-operated
(left) were triple stained with Alexa 647-conjugated a-bungarotoxin (pseudo-blue) for postsynaptic AChR, anti-synaptotagmin (SV48; red) for
presynaptic nerve terminal, and anti-PRiMA (green), and examined by confocal microscopy. Merged images allow a comparison of PRiMA
with presynaptic (SV48 + PRiMA) and postsynaptic (AChR + PRiMA) markers. The disappearance of the presynaptic nerve terminals in
denervated muscle is verified by the absence of SV48 labeling. PRiMA labeling was considerably reduced, but not completely absent. Repre-
sentative images are shown, n = 3. Bar, 10 lm.
Proline-rich membrane anchor at neuromuscular junctions K. W. Leung et al.
3038 FEBS Journal 276 (2009) 3031–3042 ª 2009 The Authors Journal compilation ª 2009 FEBS
motor neuron and the Schwann cell. During develop-
ment, the muscle is the primary source of all forms of
AChE [1]. In contrast, the contribution of the Schw-
ann cell, if any, is limited [14]; however, the possible
presence of PRiMA in the Schwann cell membrane
could only be distinguished by electron microscopy. In
this study, we confirmed the expression of PRiMA, as
well as of PRiMA-linked G
4
AChE, in the motor neu-
rons of the spinal cord using a PRiMA antibody. The

AChE, has been
revealed in brain and retina during development [21].
Our current and past results [15] indicate that motor
neurons represent the major cell type expressing
PRiMA and AChE
T
in the spinal cord. In line with
this observation, it has been shown that AChE is
expressed in both neurotube and myotomes [22]. In
addition, previous studies have also shown that AChE
synthesized in the motor neuron is transported by axo-
nal flow to the presynaptic terminal, as revealed by
enzymatic and microscopic studies [13]. The function
of pre- and postsynaptic PRiMA-linked G
4
AChE
expressed by motor neuron and muscle, particularly
during early stages of development, is an open ques-
tion. One of the proposed functions of two-sided
expression of AChE in both pre- and postsynaptic
membranes is to play an active role during synapto-
genesis through the adhesive function of AChE [23,24].
In addition, the decrease in PRiMA and AChE expres-
sion in the rat spinal cord after section of the sciatic
nerve could be the consequence of trauma or of the
loss of retrograde influence from the muscle cells.
Indeed, muscle-derived factors control the expression
of presynaptic proteins by motor neurons at NMJs
[17,25].
In previous studies, G

pLysE Escherichia coli (Invitrogen, Carlsbad, CA, USA)
and purified by glutathione bead chromatography
(Amersham Biosciences, Piscataway, NJ, USA), according
to the manufacturer’s instructions. After digestion by
thrombin (Sigma, St Louis, MO, USA), the PRiMA
(amino acids 114–153) antigen was purified by Superdex
75 10 ⁄ 300 gel filtration chromatography (Amersham
Biosciences). Polyclonal antibodies were raised in a 2-kg
male New Zealand White rabbit by immunization with
750 lg of antigen, mixed with an equal volume of
complete Freund’s adjuvant (Sigma). The immunization
was carried out with the same amount of antigen three
times within 1 month. The anti-PRiMA serum was col-
lected and purified by protein G-Sepharose (Amersham
Biosciences), according to the manufacturer’s instructions.
The amount of purified antibody was determined spectro-
photometrically.
DNA construction and transfection
The HEK293T cell line was obtained from the American
Type Culture Collection (ATCC, Manassas, VA, USA) and
K. W. Leung et al. Proline-rich membrane anchor at neuromuscular junctions
FEBS Journal 276 (2009) 3031–3042 ª 2009 The Authors Journal compilation ª 2009 FEBS 3039
cultured in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum. Cultured cells
were incubated at 37 °C in a water-saturated 5% CO
2
incu-
bator. All reagents for cell cultures were from Invitrogen.
cDNAs encoding full-length mouse PRiMA (PRiMA I) and
a COOH-terminal truncated mutant (PRiMA

bands in the control and stimulated samples, run on the
same gel and under strictly standardized enhanced chemi-
luminescence conditions, were compared on an image
analyzer using, in each case, a calibration plot constructed
from a parallel gel with serial dilutions of one of the
samples.
Immunofluorescence analysis
Transfected cell cultures or tissue sections (16 lm) were
fixed by 4% paraformaldehyde in NaCl ⁄ P
i
for 15 min,
followed by 50 mm ammonium chloride (NH
4
Cl) treatment
for 25 min. Samples were permeabilized by 0.2% Triton
X-100 in NaCl ⁄ P
i
for 10 min and blocked by 5% BSA in
NaCl ⁄ P
i
for 1 h at room temperature. Cultures were
stained with PRiMA (2 lgÆmL
)1
) or FLAG (1 : 500,
Sigma) antibodies. Tissue sections were double or triple
stained by rhodamine-conjugated or Alexa 647-conjugated
a-bungarotoxin (dilution 1 : 500; Molecular Probes,
Eugene, OR, USA), PRiMA antibody (2 lgÆmL
)1
), AChE

and centrifuged at 175 000 g in a Sorvall TH 641 rotor at
4 °C for 16 h. Approximately 45 fractions were collected
and AChE enzymatic activity was determined according to
the method of Ellman [29]; the reaction medium contained
0.1 mm tetra-isopropylpyrophosphoramide, an inhibitor of
BChE. Absorbance at 410 nm was recorded as a function
of the reaction time. The proportions of the various
AChE forms were determined by summation of the enzy-
matic activities corresponding to the peaks of the sedimen-
tation profile. In the immunoprecipitation of G
4
AChE by
PRiMA antibody, 1 mL samples of tissue extracts were
incubated for 4 h at 4 °C with purified PRiMA antibody
(10 lgÆmL
)1
). Then, 50 lL of washed protein-G agarose
gel (Santa Cruz Biotechnology) was added and incubated
for 1 h at 4 °C. After centrifugation, the supernatants
were loaded onto sucrose gradients for sedimentation
analysis.
ELISA for PRiMA-linked G
4
AChE
Fifty microliter samples of serially diluted PRiMA antibody
were coated in a 96-well ELISA plate (Nunc Maxisorp
Immunoplate, Roskilde, Denmark) for 16 h. The antibody
was removed and the plate was washed twice with 200 lL
NaCl ⁄ P
i

CGTTCCTG-3¢ for mouse AChE
T
[30]; 5¢-TGTGATGC
CCTTAGATGTCC-3¢ and 5¢-GATAGTCAAGTTCGAC
CGTC-3¢ for rat 18S ribosomal RNA. The SYBR green
signal was detected by an Mx3000pÔ multiplex quantitative
PCR machine (Stratagene, La Jolla, CA, USA). Transcript
expression levels were quantified using the DDCt value
method [31], where values were normalized to 18S rRNA
as an internal control in the same sample. PCR products
were analyzed by gel electrophoresis and the specificity of
amplification was confirmed by the melting curves.
Sciatic nerve section
Two-month-old Sprague–Dawley rats weighing approxi-
mately 250 g were anesthetized by isoflurane. A portion of
approximately 3 mm of the sciatic nerve located around
the upper thigh was removed by an aseptic surgical tech-
nique [13]. The rats were sacrificed according to the
instructions of the Animal Care Facility of The Hong
Kong University of Science and Technology. Spinal cord
(lumbar) and tibialis muscles were collected 7 days after
denervation. Samples were frozen in liquid nitrogen imme-
diately after dissection and stored at )80 °C for RNA and
protein extraction, and for confocal microscopy. Control
experiments were performed by sham operation on diff-
erent rats.
Other assays
Protein concentrations were measured by Bradford’s
method [32] with a kit from Bio-Rad Laboratories (Hercu-
les, CA, USA). Statistical tests were performed by the

5 Perrier AL, Massoulie
´
J & Krejci E (2002) PRiMA: the
membrane anchor of acetylcholinesterase in the brain.
Neuron 33, 275–285.
6 Perrier NA, Khe
´
rif S, Perrier AL, Dumas S, Mallet J &
Massoulie
´
J (2003) Expression of PRiMA in the mouse
brain: membrane anchoring and accumulation of ‘tailed’
acetylcholinesterase. Eur J Neurosci 18, 1837–1847.
7 Lyles JM, Silman I & Barnard EA (1979) Developmen-
tal changes in levels and forms of cholinesterases in
muscles of normal and dystrophic chickens. J Neuro-
chem 33, 727–738.
8 Massoulie
´
J (1993) Molecular and cellular biology of
cholinesterases. Prog Neurobiol 41, 31–91.
9 Xie HQ, Choi RCY, Leung KW, Siow NL, Kong LW,
Lau FTC, Peng HB & Tsim KWK (2007) Regulation
of a transcript encoding the proline-rich membrane
anchor of globular muscle acetylcholinesterase. The sup-
pressive roles of myogenesis and innervating nerves.
J Biol Chem 282, 11765–11775.
10 Bacou F (1982) Acetylcholinesterase forms in fast and
slow rabbit muscle. Nature 296, 661–664.
11 Koenig J & Vigny M (1978) Neural induction of the

17 Jiang JXS, Choi RCY, Siow NL, Lee HHC, Wan DCC
& Tsim KWK (2003) Muscle induces neuronal expres-
sion of acetylcholinesterase in neuron-muscle co-culture:
transcriptional regulation mediated by cAMP-dependent
signaling. J Biol Chem 278, 45435–45444.
18 McMahan UJ, Sanes JR & Marshall LM (1987)
Cholinesterase is associated with the basal lamina at
the neuromuscular junction. Nature 271 , 172–174.
19 Couraud JY & Di Giamberardino L (1980) Axonal
transport of the molecular forms of acetylcho-
linesterase in chick sciatic nerve. J Neurochem 35,
1053–1066.
20 Couraud JY, Di Giamberardino L, Hassig R & Mira
JC (1983) Axonal transport of the molecular forms of
acetylcholinesterase in developing and regenerating
peripheral nerve. Exp Neurol 80, 94–110.
21 Layer PG, Alber R & Sporns O (1987) Quantitative
development and molecular forms of acetyl- and butyr-
ylcholinesterase during morphogenesis and synaptogen-
esis of chick brain and retina. J Neurochem 49,
175–182.
22 Layer PG, Alber R & Rathjen FG (1988) Sequential
activation of butyrylcholinesterase in rostral half
somites and acetylcholinesterase in motoneurones and
myotomes preceding growth of motor axons. Develop-
ment 102, 387–396.
23 Layer PG (1990) Cholinesterases preceding major tracts
in vertebrate neurogenesis. BioEssays 12, 415–420.
24 Layer PG (1991) Expression and possible functions of
cholinesterases during chicken neurogenesis. In Cholin-

31 Winer J, Jung CK, Shackel I & Williams PM (1999)
Development and validation of real-time quantitative
reverse transcriptase-polymerase chain reaction for
monitoring gene expression in cardiac myocytes in vitro.
Anal Biochem 270, 41–49.
32 Bradford MM (1976) A rapid and sensitive method for
the quantitation of microgram quantities of protein uti-
lizing the principle of protein-dye binding. Anal Bio-
chem 72, 248–254.
33 Glantz SA (1988) Primer of Biostatistics. McGraw-Hill
Inc., NY.
Proline-rich membrane anchor at neuromuscular junctions K. W. Leung et al.
3042 FEBS Journal 276 (2009) 3031–3042 ª 2009 The Authors Journal compilation ª 2009 FEBS