Journal of Neuroscience Research 56:199–205 (1999)

Highly Basic Myelin and Oligodendrocyte

Proteins Analyzed by NEPHGE-
Two-Dimensional Gel Electrophoresis:
Recognition of Novel Developmentally
Regulated Proteins
Y. Yamaguchi and S.E. Pfeiffer*
Department of Microbiology, University of Connecticut Medical School, Farmington

Two-dimensional polyacrylamide gel electrophoresis of investigation. High resolution, two-dimensional poly-

(2D-PAGE) provides high resolution separation of acrylamide gel electrophoresis (2D-PAGE), in which
proteins and offers a powerful method for their isoelectric focusing (IEF) is carried out in the first
identification and characterization. Since many myelin- dimension followed by sodium dodecylsulfate (SDS)-
specific proteins are highly basic, they cannot readily PAGE in the second dimension (O’Farrel, 1975), affords
be analyzed by standard isoelectric focusing (IEF)- one key approach. Standard IEF-2D-PAGE resolves
2D-PAGE that affords separation primarily in the proteins with isoelectric points (pI) in the pH range 4–8.
isoelectric points (pI) range of 4–8. An alternative This technique has been coupled with a protocol for
method, nonequilibrium pH gradient electrophoresis producing populations of oligodendrocytes at specific
(NEPHGE)-2D-PAGE, can provide excellent resolu- stages of the developmental lineage (Pfeiffer et al., 1993;
tion of highly basic proteins. In the present study, we Bansal et al., 1996) to study oligodendrocyte differentia-
have optimized the NEPHGE-2D-PAGE protocol for tion. For example, Huber et al. (1994) analyzed the
the analysis of myelin proteins with basic pIs, and developmental regulation of the expression of small
provide a NEPHGE-2D-PAGE map based on size, pI, GTP-binding proteins; Kim et al. (1995) identified MVP17
and immunoreactivity (Western blot) of myelin basic (rMAL; Schaeren-Wiemers et al., 1995), a myelin protein
protein (MBP), 28,38-cyclic-nucleotide 38-phosphodies- enriched in detergent-insoluble glycosphingolipid-en-
terase (CNP), myelin proteolipid protein (PLP), and riched complexes (DIGs).
its smaller spliced variant DM20, myelin/oligodendro- However, this form of IEF-2D-PAGE poses prob-
cyte glycoprotein (MOG) and oligodendrocyte-spe- lems for the analysis of protein with pIs ⬎ 8, since such
cific protein (OSP). We have also demonstrated, by proteins become compacted at the edge of the gel. Even if
analyzing metabolically radiolabeled oligodendro- the IEF pH gradient is extended to high pH, slightly basic
cytes in culture at specific stages of the developmental proteins enter the gel but are not well resolved, and the
lineage, the developmentally up-regulated expressions pH gradient is not sufficiently extended to include highly
of several undefined, oligodendrocyte, basic mem- basic proteins (Rickwood et al., 1990). The analysis of
brane proteins during oligodendrocyte differentiation. myelin proteins offers a specific example of these prob-
We suggest that this approach offers an important tool lems. Many myelin proteins are highly basic (pI 8–12),
for identifying and characterizing the plethora of suggesting interactions with acidic sulfo- and phospholip-
uncharacterized myelin proteins. J. Neurosci. Res. ids which may promote the formation and maintenance of
56:199–205, 1999. r 1999 Wiley-Liss, Inc. compact myelin structure (Kirshner and Blaurock, 1994).
Key words: 2D-PAGE; early progenitor; oligodendro-
cyte; differentiation Contract grant sponsor: National Institutes of Health; Contract grant
number: NS10861; Contract grant sponsor: National Multiple Sclerosis
Society; Contract grant number: RG2182; Contract grant sponsor:
INTRODUCTION Japan Society for the Promotion of Science.
Although a remarkable number of proteins have *Correspondence to: Steven E. Pfeiffer, Department of Microbiology,
been characterized, the identification and characterization University of Connecticut Medical School, 263 Farmington Avenue,
of additional, often novel proteins, and the analysis of the Farmington, CT 06032–3205. E-mail: [email protected]
regulation of their expression continue to be active areas Received 28 December 1998; Accepted 8 January 1999

r 1999 Wiley-Liss, Inc.

200 Yamaguchi and Pfeiffer

A solution for the analysis of highly basic proteins NEPHGE was carried out according to a method
is found in the application of nonequilibrium pH gradient modified from that of Celis et al. (1995) and Mozdza-
electrophoresis (NEPHGE)-2D-PAGE (O’Farrel et al., nowski et al. (1995). For six tubes (3.5 mm diameter,
1977). In the first dimension, the sample is applied and 13-cm-long), the gel mixture was composed of 5.5 g urea
migrates from the positive end of the gel. Upon electropho- (ultrapure), 1.0 ml 40% acrylamide-bis (19:1), 2 ml 10%
resis (during which the pI gradient is forming), positively NP-40, 225 µl ampholyte pH 7–9, 25 µl Ampholine pH
charged, basic proteins move toward the negative end of the 9–11, 2.5 ml milliQ H2O, 20 µl 10% ammonium persul-
gel. Since running the gel to equilibrium would result in many fate, and 14 µl N,N,N8,N8-tetramethylethylenediamine
highly basic proteins exiting from the basic end of the gel (TEMED). Before polymerization, gel mixture without
(negative electrode), the electrophoresis must be stopped ammonium persulfate and TEMED were degassed under
at a critical point (thus, ‘‘nonequilibrium’’). vacuum for 5–10 min. The tube bottoms were sealed by
In this report, we first present an optimized parafilm, stood vertically, and marked at a height of 13
NEPHGE-2D-PAGE protocol for the analysis of known cm. The gel mixture with ammonium persulfate and
basic myelin proteins of the central nervous system (CNS) TEMED was poured into tubes to the 13 cm height,
and provide a NEPHGE-2D-map of their distribution. We overlaid with 50 µl 8 M urea and allowed to polymerize
then examine the developmental regulation of expression of a for ⬎2 hr. After removing the 8 M urea, the tops of gels
number of uncharacterized, highly basic, membrane proteins were equilibrated for ⬎2 min by the addition of 50 µl of
expressed by oligodendrocytes differentiating in culture. lysis buffer consisting of 2.59 g urea, 2 ml 10% NP-40,
0.5 ml 1.0 M dithiothreitol, 0.3 ml ampholyte pH 7–9, and
MATERIALS AND METHODS 0.35 ml MilliQ H2O. Proteins of myelin and cultured cell
membrane fractions were solubilized in lysis buffer. After
Materials
removing the lysis buffer overlay, solubilized samples (50
Ampholine 7–9 and Ampholyte 9–11 (Sigma Chemi- µl) were applied to the top of the NEPHGE gels and
cal Co., St. Louis, MO), acrylamide/bis-acrylamide (19:1) overlaid with 10 µl 80% lysis buffer. The lower chamber
mixture (Biorad, Hercules, CA), ultrapure grades of urea of the electrophoresis apparatus was filled with 20 mM
and dithiothreitol and prestained marker mixtures for NaOH, and the upper chamber was filled with 10 mM
SDS-PAGE (Gibco, Grand Island, NY) were used. Poly- H3PO4. The electrode from the upper chamber was
clonal antibodies to MBP and CNP were prepared in our connected to the (⫹) socket of a power supply, and the
laboratory immunizing rabbits against purified myelin electrode from the lower chamber was connected to the
basic protein (MBP) and 28,38-cyclic-nucleotide 38- (⫺) socket. Electrophoresis was run at 600 V for 2.5 hr
phosphodiesterase (CNP). Hybridoma cells producing (see Results). After electrophoresis, each gel was trans-
anti-myelin proteolipid protein (PLP)/DM20 monoclonal ferred from its tube to a 60 mm diameter plastic petri dish
antibody (AA3) were derived by Dr. Kazuhiro Ikenaka and incubated for 15 min at room temperature in 6 ml
from an original line developed by Dr. Majorie B. Lees equilibrium buffer (pH 8.65) containing 3% SDS, 0.4 mM
and associates. Polyclonal antibodies, anti-myelin/ ethylenediaminetetraacetic acid (EDTA), 10% glycerol,
oligodendrocyte glycoprotein (MOG) and anti-oligoden- and 20 mM Tris-HCl. Most of the liquid was then
drocyte-specific protein (OSP) were kindly provided by removed and the gels were quickly frozen in the petri
Drs. Claude C. Bernard (La Trobe University, Australia) dishes on dry ice, wrapped in parafilm, and stored at
and Jeff M. Bronstein (UCLA), respectively. All solutions ⫺80°C.
were prepared with MilliQ H2O. Protein concentrations In the second dimension, SDS-PAGE (5–20% gradi-
were determined by DC protein assay kit (Bio-Rad,Rich- ent gel, 16 cm x 16 cm, 1-mm-thick) was carried out
mond, CA). using the methods of Laemmli (1970) and Mozdzanowski
et al. (1995).
Purification of Rat Brain Myelin
Myelin was purified from adult rats using previ- Silver Stain
ously described methods (Huber et al., 1994; Kim et al., Silver stain was carried out according to a method
1995) based on those of Norton and Poduslo (1973), modified from that of Merril et al. (1994). Gels were
Fujimoto et al. (1976), and Haley et al. (1981). stained with 0.1% Coomassie brilliant blue in 50%
methanol/10% acetic acid for 30 min and destained
Two-Dimensional Polyacrylamide Gel overnight in 10% methanol/10% acetic acid. Destained
Electrophoresis (2D-PAGE) gels were soaked in MilliQ H2O for 5 min, incubated in
Standard IEF gel electrophoresis in the first dimen- 2% glutaraldehyde for 10 min and washed 5 times in
sion was carried out as described previously (Wandinger- MilliQ H2O (two times briefly, three times for 10 min
Ness et al., 1990; Kim et al., 1995). each). The gels were then incubated for 15 min in 100 ml
 NEPHGE-2D-PAGE of Myelin Proteins 201

ammoniacal silver solution containing 5 ml 15% silver TABLE I. Theoretical Isoelectric Points (pIs) and Molecular
nitrate and 5 ml of 0.15% NaOH/1% NH4OH, washed Weights of Some Known Myelin Proteins*
five times in MilliQ H2O (two times briefly, three times Molecular
for 5 min each) and developed for a few minutes in a weight
solution (100 ml) containing 0.05% citrate, 0.5% formal- pI (kDa)
dehyde. Staining was terminated by adding 2.5 ml of 2.3 MBP(14K) (mouse) 11.8 14.1
M citrate to the developing solution and incubating the MBP(17K) (mouse) 11.8 17.1
gels for 5 min. The gels were then washed three times for MBP(18.5K) (mouse) 11.1 18.4
MBP(21.5K) (mouse) 11.2 21.4
10 min each in MilliQ H2O. CNP I (rat) 8.6 44.7
CNP II (rat) 9.0 47.1
PLP (rat) 8.7 29.9
Western Blot DM20 (rat) 8.2 26.1
After electrophoresis, proteins were transferred at MOBP (mouse) 9.2 8.2
100 mA for 1 hr to nitrocellulose membrane in a solution MOG (rat) 8.6 27.8
mOMGP (mouse) 8.7 49.2
containing 10% methanol, 20 mM Tris, and 100 mM OSP (mouse) 8.2 22.0
glycine. The membrane was blocked for 1 hr in a solution Plasmolipin (rat) 9.3 17.3
containing 5% skim milk, 0.1% Tween 20, 0.15 M NaCl, Connexin 32 (rat) 9.2 32.0
and 20 mM Tris-HCl (pH 7.4). Proteins were detected by MAG-L (rat) 5.0 69.2
the ECL system (Amersham, Arlington Heights, IL) using MAG-S (rat) 4.9 64.2
antibodies specific for the basic myelin proteins anti- MVP17/rMAL (rat) 5.7 16.6
MBP (1/20,000), anti-CNP (1/10,000), AA3 (1/1,000), *Theoretical pIs and molecular weights were calculated using a
anti-MOG (1/10,000), or anti-OSP (1/5,000). program in the ExPASy Molecular Biology server of the Swiss Institute
of Bioinformatics (SIB) (http://expasy.hcuge.ch/). For these calcula-
tions, amino-terminal methionines were excluded. Translated amino
Preparation of Highly Enriched Oligodendrocyte acid sequences were acquired from the National Center for Biotechnol-
Cultures ogy Information (NCBI) server (http://www.ncbi.nlm.nih.gov/). MBP,
myelin basic protein; CNP, 28,38-cyclic-nucleotide 38-phosphodiester-
Mixed primary cultures and highly enriched popula- ase; PLP, myelin proteolipid protein; DM20, spliced variant of PLP;
tions of early progenitor cells and maturing oligodendro- MOBP, myelin-associated oligodendrocytic basic protein; MOG, my-
cytes (Pfeiffer et al., 1993) were prepared, characterized, elin/oligodendrocyte glycoprotein; mOMGP, mouse oligodendrocyte-
and maintained as previously described (Bansal et al., myelin glycoprotein; OSP, oligodendrocyte specific protein; MAG-(L,
S): myelin-associated glycoprotein (long or short form); MVP17/
1996; Madison et al., 1998). rMAL, myelin vesicular protein MVP17/rat myelin and lymphocyte
protein.
In Vitro Labeling of Oligodendrocytes With
[35S]Met/Cys and Preparation of Membrane
Fractions the wash buffer and cells were collected and added to the
Early progenitor cells and maturing oligodendro- original suspension (⬃300 µl/dish final volume). The
cytes in culture (2–3 ⫻ 106 cells/100 mm dish) were cells were homogenized by 10 passages through a 25G
metabolically labeled as previously described (Kim et al., needle attached to a 1 ml syringe. The homogenate was
1995) by incubating the cells in 0.7 mCi/dish of centrifuged at 1,000 ⫻ g for 10 min to remove nuclei. The
Expre35S35S (Amersham; 0.5 mCi/dish of [35S]Met) for 9 supernatant fraction (50 µl) was further centrifuged at
hr (early progenitors) or 15 hr (maturing oligodendro- 200,000 x g for 1 hr, the supernatant removed, and the
cytes). The labeled cells were washed once with ice cold precipitate solubilized directly in 50 µl lysis buffer and
Hepes-Earl’s balanced solution and twice with ice-cold stored at -80°C. The radiolabeled membrane proteins
isotonic buffer A containing 10 mM Hepes-NaOH (pH (applied amount: 1–2 x 106 dpm in 50 µl lysis buffer) were
7.4), 0.15 M NaCl, 1 mM ethylene glycol-bis(b- analyzed by NEPHGE-2D-PAGE and detected by fluorog-
aminoethyl ether)-N,N,N8,N8-tetraacetic acid (EGTA), raphy using Amplify (Amersham).
0.1 mM MgCl2, 1 mM dithiothreitol, and proteinase
inhibitors (1 mM phenylmethyl-sulfonylfluoride, 1 µg/ml
pepstatin A, 1 µg/ml antipain, 10 µg/ml aprotinin). After
RESULTS
removing nearly all of the wash solution, but avoiding
drying, the cells were collected at the corner of the dish Analysis of Known Basic Myelin Proteins
using a plastic cell scraper, and transferred to screw- Table I shows the theoretical pIs and molecular
capped 1.5 ml microcentrifuge tubes. The dishes were weights of a number of CNS myelin proteins, many of
then washed once with 50 µl/dish isotonic buffer A, and which are highly basic (pI 8–12). Analysis of myelin
202 Yamaguchi and Pfeiffer

Fig. 1. Comparison of the protein profiles of adult rat central nervous system (CNS) myelin (78
µg protein) obtained by using nonequilibrium pH gradient electrophoresis-two-dimensional
polyacrylamide gel electrophoresis (NEPHGE-2D-PAGE; left) or isoelectric focusing (IEF)-2D-
PAGE (right). Proteins are detected by silver stain. Molecular weights of marker proteins are
indicated at both sides.

proteins using standard, equilibrium IEF-2D-PAGE re- panels, box B: spot 5 [CNP II], spot 6 [CNP I]), with the
sulted, as expected, in the stacking or loss of most of these smaller, more acidic spot (CNP I) predominating. Anti-
basic proteins at the basic end (Fig. 1, right panel). In PLP monoclonal antibody recognized PLP and its spliced
contrast, the nonequilibrium NEPHGE-2D-PAGE method variant DM20 (Fig. 2, left panels, box C-1: spots 7 and 8,
allowed the resolution of these proteins (Fig. 1, left respectively). These highly hydrophobic proteolipid pro-
panel). Since NEPHGE is stopped before equilibrium is teins can oligomerize (Fig. 2, box C-1, arrows). Anti-
attained, accurate determination of the pIs of proteins MOG and -OSP polyclonal antibodies recognized pro-
cannot be made by this method. teins at the expected molecular weight (Fig. 2, boxes C-2:
Carefully regulated running conditions, controlled spot 9 and C-3: spot 10, respectively).
by a consideration of the total number of volt-hours Several proteins exhibited lateral streaking, suggest-
(V-hr), are required for successful NEPHGE analyses. ing post-translational modifications. For example, PLP
For myelin proteins, we found that 1,500 V-hr provided and OSP (Fig. 2, left panels, boxes C-1 and C-3,
optimal resolution while preventing very highly basic respectively) are likely to be acylated, while MOG (Fig.
proteins, such as MBPs, from running off the basic end of 2, left panels, box C-2) may be variably glycosylated.
the gel. A comparison of the 2D protein patterns obtained Arrowheads (Fig. 2, left panels, boxes B and C-1–3)
at constant voltages of 400 V and 600 V demonstrated a indicate immunoreactive spots at the extreme acidic end
better resolution at 600 V (data not shown). Thus, in of the gel that may be caused by their aggregation during
subsequent experiments, we ran NEPHGE gels at 600 V the first-dimension NEPHGE, thereby precluding their
for 2.5 hr (1,500 V-hr) at room temperature (20–25°C). entry into the second-dimension gel. At higher levels of
To prepare a NEPHGE-2D-PAGE map of known loading, a rather generalized background staining was
basic myelin proteins, we performed Western blot analy- observed below about pI 8.5. Although this is rarely a
ses of purified adult rat CNS myelin, using antibodies problem for most studies, it could in principle pose some
against MBP, CNP, PLP (DM20), MOG, and OSP (Fig. problems for amino acid microsequence analysis of
2). Anti-MBP antibody detected four MBP isoforms (Fig. eluted proteins. Thus, in such cases, further purification
2, left panels, box A: spot 1 [21.5K], spot 2 [18.5K], spot prior to electrophoresis may be warranted; anecdotally,
3 [17K], spot 4 [14K]); the more acidic immunoreactive we have observed that prior chloroform-methanol extrac-
spots (arrows) may represent post-translational modifica- tion reduces this background.
tions (e.g., phosphorylation); a higher molecular weight, Although the first-dimension NEPHGE separation
more acidic immunoreactive spot (arrowhead) may be is based on pI, under the nonequilibrium conditions, both
Golli-mbp (Campagnoni et al., 1993). Anti-CNP poly- pI and molecular weight affect the movement of proteins.
clonal antibody recognized two isoforms (Fig. 2, left In general, smaller and more basic molecules tend to
 NEPHGE-2D-PAGE of Myelin Proteins 203

Fig. 2. Western blot analysis of CNS myelin protein using proteolipid protein (PLP); spot 8, DM20; arrows, PLP/DM20
NEPHGE-2D-PAGE. Upper left: Protein profile of adult rat oligomers. C-2: Spot 9, myelin/oligodendrocyte glycoprotein
CNS myelin (the same as Fig. 1, left panel). Boxes A–C show (MOG). C-3: Spot 10, oligodendrocyte-specific protein (OSP).
the nitrocellulose membrane regions used for Western blots. In B and C1–3, arrowheads indicate immunoreactive spots at
Lower left: Western blots of various basic myelin proteins. the acidic end of the gel. Upper right: Composite image;
Signals are visualized by the ECL detection system. A: Spots Western blot reactivities (lower left panel) have been overlaid
1–4, myelin basic protein (MBP) isoforms; arrows and arrow- on the silver stain profile (upper left panel). Lower right:
head, MBP-related molecules. B: Spots 5, 6, 28,38-cyclic- NEPHGE-2D-PAGE map of adult rat CNS myelin proteins.
nucleotide 38-phosphodiesterase (CNP) isoforms. C-1: Spot 7, Arrows indicate the anti-MBP immunoreactive spots (box A).

move faster in NEPHGE. For example, DM20 (spot 8) ined. Early progenitor cells (EP) and maturing oligoden-
and OSP (spot 10) have similar pIs (8.2), and the smaller drocytes (mOL) were metabolically labeled with [35S]
OSP migrates slightly faster. However, exceptions are Met/Cys, and the radioactive proteins were resolved by
noted, at least relative to theoretical pI determinations. NEPHGE-2D-PAGE (Fig. 3). Several proteins were up-
For example, in the case of CNP isoform resolution, pI regulated in mOLs. By comparison of the position of
predominates: the more basic CNPII (spot 5; pI 9.0, 47.1 these proteins with the NEPHGE-2D-PAGE map of
kD) moves faster than CNPI (spot 6; pI 8.6, 44.7 kD). known CNS myelin proteins (Fig. 2, right panels), we
Similarly, the more basic PLP (spot 7; pI 8.7, 29.9 kD) could identify PLP (spot 1), DM20 (spot 2), CNP I (spot
migrates more rapidly than DM20 (spot 8; pI 8.2, 26.1 3), and MOG (spot 4; the major MBP isoforms 14, 17,
kD).
and 18.5 kD, have fewer than three residues of Met/Cys,
and therefore could not be detected by [35S]-labeling in
Expression of Developmentally Regulated, this study). At least eight of the up-regulated proteins
Oligodendrocyte Membrane Proteins (Fig. 3, arrows) appear to be novel species that are
The developmental regulation of the expression of presumably related to terminal differentiation of oligoden-
oligodendrocyte basic membrane proteins was next exam- drocytes in either a structural or regulatory capacity. In
204 Yamaguchi and Pfeiffer

Fig. 3. NEPHGE-2D-PAGE fluorogram of [35S] Met/Cys-labeled membrane proteins of early

progenitor cells (EP) and maturing oligodendrocytes (mOL). Several proteins whose expression
was up-regulated as the cells entered terminal differentiation were identified according to their
positions (1, PLP; 2, DM20; 3, CNP; 4, MOG). Up-regulated proteins which are unidentified
are indicated by arrows. Down-regulated proteins which are unidentified are indicated by
arrowheads. The numbers at left show positions of marker proteins.

addition, at least four proteins (Fig. 3, arrowheads) were Second, there are a number of known post-
present in EPs but absent from mOLs. We conclude that translational modifications of myelin proteins (e.g., phos-
this approach offers a means to identify a new set of basic, phorylation of MBP); NEPHGE-2D-PAGE has sufficient
developmentally regulated, oligodendrocyte/myelin pro- resolution to separate these proteins, and could be used to
teins. analyze protein phosphorylation in response to extracellu-
lar factors.
Third, several apparently novel oligodendrocyte
DISCUSSION proteins whose expression is regulated during oligoden-
We have developed a NEPHGE-2D-PAGE protocol drocyte differentiation could be noted by NEPHGE-2D-
for the analysis of basic CNS myelin proteins, and using PAGE analysis of [35S]-labeled cultures of oligodendro-
this procedure, established a NEPHGE-2D-PAGE map of cytes at specific stages of the developmental lineage.
these proteins. There are several advantages to analyzing Thus, NEPHGE-2D-PAGE offers a promising approach
basic myelin proteins using NEPHGE-2D-PAGE. for the discovery of basic oligodendrocyte/myelin pro-
First, while one-dimensional SDS-PAGE has been teins, using the strategy similar to that used to identify
used successfully for analyzing myelin proteins from a MVP17 (Kim et al., 1995) and oligodendrocyte small
variety of sources including mutant, transgenic, and GTP-binding proteins (Huber et al., 1994).
knockout mice (e.g., Sorg et al., 1986; Mastronardi et al.,
1993; Klugmann et al., 1997), it is limited in its resolution
for the separation of proteins of similar molecular weight. ACKNOWLEDGMENTS
NEPHGE-2D-PAGE offers, therefore, a significant en- We thank Drs. Kazuhiro Ikenaka and Majorie Lees
hancement of resolution. For example, Jensen and Celis for the AA3-hybridoma, Dr. Claude Bernard for MOG
(1998) have recently demonstrated the down-regulation antisera, and Dr. Jeff Bronstein for OSP antisera. We also
of expression of MBP and CNP in transgenic mice thank members of our laboratory, Rashmi Bansal, Win-
overexpressing c-myc under the control of a MBP fried Krueger, Taeyoon Kim, and Brian Bouverat, for
promoter. valuable discussions.
 NEPHGE-2D-PAGE of Myelin Proteins 205

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