Syndecan-4 is a signaling molecule for stromal cell-derived
factor-1 (SDF-1)
/
CXCL12
Nathalie Charnaux
1,2
*, Se
´
verine Brule
1,2
*, Morgan Hamon
1
, Thomas Chaigneau
1
, Line Saffar
1
,
Catherine Prost
1
, Nicole Lievre
1
and Liliane Gattegno
1,2
1 Laboratoire de Biologie Cellulaire, Biothe
´
rapies Be
´
ne
´
fices et Risques, UPRES 3410 Universite
´

France, Ho
ˆ
pital Jean Verdier, 93017, Bondy,
France
Fax: +33 1 48026503
Tel: +33 1 48387752
E-mail:
*These authors contributed equally to this
work.
(Received 18 January 2005, accepted 21
February 2005)
doi:10.1111/j.1742-4658.2005.04624.x
Stromal cell-derived factor-1 (SDF-1) ⁄ CXCL12, the ligand for CXCR4,
induces signal transduction. We previously showed that CXCL12 binds to
high- and low-affinity sites expressed by primary cells and cell lines, and
forms complexes with CXCR4 as expected and also with a proteoglycan,
syndecan-4, but does not form complexes with syndecan-1, syndecan-2,
CD44 or beta-glycan. We also demonstrated the occurrence of a CXCL12-
independent heteromeric complex between CXCR4 and syndecan-4.
However, our data ruled out the glycosaminoglycan-dependent binding of
CXCL12 to HeLa cells facilitating the binding of this chemokine to
CXCR4. Here, we demonstrate that CXCL12 directly binds to syndecan-4
in a glycosaminoglycan-dependent manner. We show that upon stimulation
of HeLa cells by CXCL12, CXCR4 becomes tyrosine phosphorylated as
expected, while syndecan-4 (but not syndecan-1, syndecan-2 or beta-glycan)
also undergoes such tyrosine phosphorylation. Moreover, tyrosine-phos-
phorylated syndecan-4 from CXCL12-stimulated HeLa cells physically
coassociates with tyrosine phosphorylated CXCR4. Pretreatment of the
cells with heparitinases I and III prevented the tyrosine phosphorylation of
syndecan-4, which suggests that the heparan sulfate-dependent binding of

expected, and also SD-4 [17], but not SD-1, SD-2, beta-
glycan or CD44 ([17] and unpublished data). Moreover,
we recently demonstrated the occurrence of an SDF-1-
independent heteromeric complex on the plasma mem-
brane of these cells, which comprises CXCR4 and SD-4
but not SD-1, SD-2, CD44 or beta-glycan [17]. This
suggested that SDF-1 may bind both the PG SD-4 and
its G-protein-coupled receptor (GPCR), CXCR4. How-
ever, our previous data have shown that while glycos-
aminidases pretreatment of primary macrophages
decreases the binding of SDF-1 to CXCR4, such treat-
ment had no effect on the chemokine binding to
CXCR4 expressed by the HeLa cell line [17]. This has
suggested that while SD-4 may serve as a binding
anchor for SDF-1 on primary macrophages to enable
the chemokine to interact with CXCR4, this was not
true if HeLa cells were tested.
The present study was designed to test whether
SD-4 functions as a specific SDF-1 signaling molecule.
Therefore, we first determined whether SDF-1 directly
binds SD-4 and the glycosaminoglycan (GAG)-
dependency of this binding. Because protein phos-
phorylation plays a critical role in the generation of
intracellular signals in response to external stimuli,
we then investigated whether SD-4 becomes tyrosine
phosphorylated (Ptyr) upon SDF-1 stimulation of
HeLa cells, and whether, in these conditions, tyrosine-
phosphorylated SD-4 is physically coassociated with
tyrosine-phosphorylated CXCR4, and what the GAG-
dependency of these events is. Finally, we asked whe-

heparitinases I, III, and chondroitinase ABC mixture, electroblotted
and revealed with 3G10 mAb (lane 1) or the isotype, IgG2b (lane
2). The respective immunoreactivity with anti-(SD-1) DL-101, anti-
(SD-4) 5G9, anti-CD44 mAbs, or anti-(SD-2) Igs are represented by
arrows. Data are representative of three individual experiments.
Syndecan-4 is an auxiliary receptor for SDF-1/CXCL12 N. Charnaux et al.
1938 FEBS Journal 272 (2005) 1937–1951 ª 2005 FEBS
labeling. The core proteins of most PGs enriched from
HeLa cells lysates were analyzed [17] after heparitinase
I and III and chondroitinase ABC treatment to detect
their apparent molecular masses. Proteins of 32 kDa
and 50–58 kDa, immunoreactive with anti-SD-4 5G9
and 3G10 mAbs, were observed (Fig. 1B). The 50–
58 kDa proteins may represent, in accordance with
other studies, homo- or hetero-oligomers of the SD-4
core protein, which is a 32 kDa protein [19]. Other
PGs were also detected: 34 kDa proteins immunoreac-
tive with both anti-SD-2 mAbs and mAb 3G10,
45- and 90 kDa proteins immunoreactive with anti-
SD-1DL-101 and 3G10 mAbs (the 90 kDa ones
probably being dimers of the 45 kDa ones), and
60 kDa proteins immunoreactive with anti-CD44 and
3G10 mAbs (Fig. 1B). All these apparent molecular
masses are close to the predicted ones [9]. These PGs
were glycanated, as mAb 3G10 reacts with an epitope
including a terminal unsaturated uronic acid residue,
which is unmasked after HS removal by heparitinases
treatment [20].
Native PGs may migrate in a diffuse high molecular
mass distribution on SDS ⁄ PAGE. Using the respective

was further analyzed by electron microscopy (Fig. 3B).
Beads at the cell surface were counted and considered
as associated when the distance between them was less
than 15 nm. Forty per cent of the beads that labeled
SD-4 were associated with 45% of the beads that labe-
led SDF-1a, while no association of SDF-1a with
SD-1 was detected. Controls, run without biotinylated
SDF-1a or with the isotypes, were not stained (data
not shown).
Fig. 2. SDF-1 binds to SD-4. HeLa cells were lysed in the presence of Triton X-100 and urea. PGs were enriched by DEAE Sephacel anion
exchange chromatography, electroblotted and revealed with anti-SD-4 5G9 mAb (lane 1), anti-(SD-1) DL-101 mAb (lane 2), anti-CD44 mAb
(lane 3), anti-(SD-2) Igs (lane 4), anti-(beta-glycan) Igs (lane 5), anti-HS 10E4 mAb (lane 6), biotinylated SDF-1a (lane 7). Alternatively, strips
were treated with heparitinases I, III mixture and revealed with anti-HS mAb 10E4 (lane 8) or biotinylated SDF-1a (lane 9). Data are represen-
tative of three individual experiments.
N. Charnaux et al. Syndecan-4 is an auxiliary receptor for SDF-1/CXCL12
FEBS Journal 272 (2005) 1937–1951 ª 2005 FEBS 1939
SDF-1 induces the tyrosine phosphorylation of
CXCR4 and the homo- or hetero-oligomerization
of this GPCR on HeLa cells
SDF-1-activated or nonactivated HeLa cell lysates
were either immunoprecipitated with anti-CXCR4 mAb
G19 then blots were developed with anti-Ptyr mAb
4G10, or immunoprecipitated with anti-Ptyr mAb 4G10
then blots were developed with anti-CXCR4 mAb
12G5. A protein was observed at 48 kDa, and several
others of apparent molecular masses > 48 kDa. All
amounts of these tyrosine-phosphorylated proteins
were significantly increased upon SDF-1 stimulation
of the cells (Fig. 4A, lane 2 vs. 1, and lane 4 vs. 3).
These increases were not significant if the cells were

precipitated in parallel with anti-Ptyr mAb 4G10 and
blots were developed with several different anti-PG Abs:
anti-SD-4 mAb 5G9, anti-SD-1 mAb DL-101, anti-SD-
2 Abs or anti-beta-glycan Abs (Fig. 4B and data not
shown). The tyrosine phosphorylated smear described
above was only significantly observed when the anti-
Ptyr 4G10 IP from SDF-1-activated HeLa cell lysates
Syndecan-4 SDF-1α
Merged
A
B
Fig. 3. SDF-1 colocalizes with SD-4 on HeLa
cells. (A) HeLa cells were double stained
with fluorescently labeled biotinylated
SDF-1a (green) and anti-(SD-4) mAb 5G9
(red). Confocal microscopy analysis shows
the colocalization of biotinylated SDF-1a
with SD-4, as assessed by the yellow
(red-green) colocalization, suggesting the
clustering of SDF-1 and SD-4. Data are
representative of three individual experi-
ments. Bar ¼ 5 lm. (B) HeLa cells were
double-stained with biotinylated SDF-1a and
with anti-(SD-4) mAb. Stainings were
revealed with streptavidin-15 nm colloidal
gold particles or anti-mouse Ig bound to
6 nm colloidal gold particles, respectively.
Black arrows show colocalization of 6- and
15-nm colloidal gold particles. Bar ¼ 100 nm
(initial magnification · 27 500). Data are rep-

The protein core of tyrosine phosphorylated SD-4
was examined in parallel after digestion of the GAGs
chains (Fig. 5B). For this purpose, the anti-Ptyr 4G10
IPs and the anti-SD-4 5G9 IPs of the SDF-1-unstimu-
lated and stimulated HeLa cells were treated with a
AB
D
C
Fig. 4. SDF-1 induces the tyrosine-phosphorylation of SD-4 on HeLa cells. Confluent serum-starved HeLa cells were either stimulated (+) or
not (–) with SDF-1a. Equal amounts of proteins from whole cell extracts were immunoprecipitated with the indicated antibodies and equival-
ent amounts of IP samples were separated on 12% SDS ⁄ PAGE and immunoblotted using the indicated mAb or polyclonal antibodies. (A)
HeLa cells were stimulated (+) (lanes 2,4) or not (–) (lanes 1,3) for 10 min with 125 n
M SDF-1a. Cell lysates were immunoprecipitated either
with anti-CXCR4 Igs G19 (lanes 1,2) or anti-Ptyr mAb 4G10 (lanes 3,4). Western blots were developed, respectively, with anti-Ptyr mAb
4G10 (lanes 1,2) or anti-CXCR4 mAb 12G5 (lanes 3,4). (B) HeLa cells were stimulated (+) (lanes 2,3,4,6,8) or not (–) (lanes 1,5,7) with
125 n
M SDF-1a for the indicated time. Cell lysates were immunoprecipitated with anti-Ptyr mAb 4G10 (lanes 1–8). Western blots were
developed with anti-(SD-4) mAb 5G9 (lanes 1–4), anti-b-glycan Abs (lanes 5,6) or anti-(SD-2) Igs (lanes 7,8). (C,D) The intensities of the phos-
phorylated bands shown in A and B (lanes 1–4) were quantified in absorbance units by densitometric scanning and analyzed with
SCION IMAG-
ER
. They were expressed as ratios of the data observed for the SDF-1 stimulated cells relative to the untreated control cells. Each bar
represents the mean ± SE of triplicate determinations of an individual experiment. The significance of the differences as compared with
untreated control cells was assessed using Student’s t-test: **P < 0.05. The position of immunoglobulin chains is indicated by a star. Pro-
tein bands with changes in tyrosine phosphorylation state are indicated by arrows.
N. Charnaux et al. Syndecan-4 is an auxiliary receptor for SDF-1/CXCL12
FEBS Journal 272 (2005) 1937–1951 ª 2005 FEBS 1941
B
A
C

glycan) Igs (Fig. 5B and data not shown).
We then used coimmunoprecipitation experiments to
further analyse the physical association of CXCR4 and
SD-4. The anti-SD-4 5G9 IPs of unstimulated as well
as SDF-1-stimulated HeLa cells lysates, respectively,
were characterized by the presence of 48 kDa proteins
and of several other minor proteins of apparent
molecular masses > 48 kDa, all immunoreactive with
12G5 (Fig. 5C, lanes 1 and 2). Therefore, the SDF-1-
independent heteromeric complex between CXCR4 and
SD-4 (Fig. 5C, lane 1) is still present if the cells are sti-
mulated by the chemokine (Fig. 5C, lane 2 vs. 1).
The tyrosine phosphorylation of SD-4 induced
by SDF-1 on HeLa cells depends on the HS chains
of this PG
To examine whether the tyrosine phosphorylation of
SD-4 induced by SDF-1 on HeLa cells depends on
HS, we treated these cells with mixtures of heparitinase
I and III prior to their stimulation by SDF-1. To pre-
serve cell viability, concentrations of heparitinases were
lower than those used to treat the IPs. The efficiency
of the enzymes was investigated: if the cells were incu-
bated in enzyme-free medium and then stimulated with
SDF-1, the 5G9 IPs revealed with 10E4 showed, as
expected, the 110–200 kDa broad smear, described
above; however, if the cells were pretreated with hepari-
tinases I and III, this smear was no longer present
(data not shown). Moreover, this heparitinases pre-
treatment of the cells prevented in a significant manner
the tyrosine-phosphorylation of SD-4 induced by

out nonspecific protein association with membrane
components under our experimental conditions.
The activation of p44
/
p42 MAPK and JNK
/
SAP
kinase by SDF-1 on HeLa cells is HS- and
SD-4- dependent
To analyze some of the transduction pathways induced
by SDF-1 on HeLa cells, whole cell extracts from
either unstimulated or stimulated HeLa cells were elec-
troblotted and revealed using phospho-specific anti-
p44 ⁄ p42 mitogen-activated protein kinase (MAPK) or
anti-p46 ⁄ p54-Jun N-terminal ⁄ stress-activated protein
kinase (JNK ⁄ SAP kinase) Abs, respectively. Parallel
immunoblottings with anti-total polyclonal Abs
confirmed equal loading of the samples (Fig. 6). As
expected [24–26], SDF-1a and phorbol 12-myristate-
13-acetate (PMA) induced a rapid activation of p44 ⁄ 42
MAPK and JNK ⁄ SAP kinase signaling in HeLa cells
by increasing phosphorylations of the respective pro-
teins (Fig. 6). This effect was time and concentration-
dependent: It rose from 3 nm up to 125 nm SDF-1a
and if the time of incubation with the chemokine was
enhanced from 5 to 15 min. On the contrary, these
phosphorylations decreased if the time of incubation
with the chemokine was enhanced up to 30 min
(Fig. 6A). According to these results, the cells were
incubated for 15 min in the presence of 125 nm of

100
0
phosphorylation level
(% versus control)
phosphorylation level
(% versus control)
**
**
A
B
Fig. 6. Heparan sulfate is involved in the activation of MAPK induced by SDF-1 stimulation of HeLa cells. (A) Serum-starved HeLa cells were
either stimulated or not with 3 n
M or 125 nM SDF-1a for 5, 15 and 30 min, and then analyzed for p44 ⁄ p42 MAPK and JUN ⁄ SAPK activations.
(B) Upper panel: Untreated (–) or heparitinases I- and III-treated (+) HeLa cells were either stimulated or not for 10 min with PMA (0.5 l
M)or
SDF-1a (125 n
M).Whole cell extracts were separated on 12% SDS ⁄ PAGE and immunoblotted using either phosphospecific anti-(p44 ⁄ p42
MAPK) or phosphospecific p46 ⁄ p54-SAPK ⁄ JNK rabbit polyclonal antibodies. Parallel immunoblottings with anti-(total p44 ⁄ p42 MAPK) or anti-
(total p46 ⁄ p54-SAPK ⁄ JNK) polyclonal antibodies, respectively, confirmed equal loading of samples. Lower panel in (B): The results were
quantified by densitometric scanning and analyzed with
SCION IMAGER. For each lane, data were expressed as p44 ⁄ p42 or SAPK ⁄ JNK phos-
phorylated proteins over total proteins in absorbance units. The amount of MAPK (p44 ⁄ p42 or SAPK ⁄ JNK) phosphorylation in the SDF-1-sti-
mulated cells was calculated according to the level of phosphorylated MAPK proteins in unstimulated control cells, which was considered as
100%. Each bar represents the mean ± SE of triplicate determination of an individual experiment. The significance of the differences
between the SDF-1-stimulated cells and the corresponding heparitinases treated cells was assessed using a t -test. **P < 0.05.
Syndecan-4 is an auxiliary receptor for SDF-1/CXCL12 N. Charnaux et al.
1944 FEBS Journal 272 (2005) 1937–1951 ª 2005 FEBS
(Fig. 8B–D and data not shown). To monitor
the sequence specificity for SD-4 RNA interference,
mutSD-4 dsRNAs was used as a control. The mutSD-

detected strongly argues for the selectivity of this bind-
ing. We then examined whether SDF-1 is associated
with SD-4 at the plasma membranes of intact HeLa
cells. By using both confocal and electron microscopy
analysis, we show strong evidence for the occurrence
of a colocalization between SDF-1 and SD-4 at the
HeLa cell plasma membrane. The fact that in the same
conditions, no colocalization of SDF-1 with another
PG, SD-1, was observed, argues further for the selec-
tivity of this association. Therefore, our findings
observed at the molecular level were strengthened by
experiments performed at the cellular level.
Thereafter, we asked whether GAGs are involved in
SDF-1 binding to SD-4. By pretreating the electroblot-
ted PGs from the HeLa cells with heparitinase I and
III and chondroitinase ABC mixture, we demonstrate
the strong GAG dependency of this binding. However,
our data do not rule out the additional involvement of
protein–protein interactions between SDF-1 and the
SD-4 core protein. Indeed, while the SD core proteins
share a high degree of conservation in their short cyto-
plasmic and transmembrane domains, in contrast their
120
60
0
120
60
0
050100
0 50 100

SD-4 dsRNA transfected HeLa cells (D) were
loaded for 30 min with Fluo-3 and then
stimulated with SDF-1a (125 n
M), as indica-
ted by black arrows. The plots show the
variations of the fluorescence intensity
(expressed in arbitrary units), measured
overtime within the analyzed cells. Data
are representative of three individual
experiments.
N. Charnaux et al. Syndecan-4 is an auxiliary receptor for SDF-1/CXCL12
FEBS Journal 272 (2005) 1937–1951 ª 2005 FEBS 1945
extracellular domains are divergent with the exception
of consensus sites for GAG attachment [15,35].
The participation of the SD-4 ectoplasmic domain in
SDF-1 binding raises the question whether this binding
is accompanied by intracellular modifications of SD-4
such as tyrosine phosphorylation, which plays critical
role in a variety of cellular processes. We have therefore
asked whether SD-4 functions as an SDF-1 signaling
molecule. For this purpose, we investigated whether
SDF-1 stimulation of HeLa cells induces an increase in
the tyrosine phosphorylation of SD-4, besides that of
CXCR4 which has already been reported [21–23]. The
SD cytoplasmic domains contain four conserved tyro-
sine residues, two of which are in favorable sequences
for phosphorylation [36]. Endogenous tyrosine phos-
phorylation of SDs has already been detected while
most cell surface SDs are phosphorylated following
treatment with the tyrosine phosphatase inhibitor per-

0
32
10
1
10
2
10
3
10
0
10
1
10
2
10
3
10
4
IgGl
IgGl
(SD-4ds RNA)
SD-1 (SD-4 ds RNA)
SD-1 (mocktransfected)
(mocktransfected)
Fig. 8. SD-4 is involved in SDF-1 activation of MAPK pathways. HeLa cells were transfected with either SD-4 dsRNAs or MutSD-4 dsRNA or
were mock-transfected. (A) Left panel: HeLa cells were analyzed for SD-4, SD-1, SD-2, CXCR4 specific mRNA, by semiquantitative RT-PCR,
3 days post transfection. To normalize for input of total RNA, GAPDH mRNA was also determined. Right panel: SD-4 mRNA levels were
quantified by densitometric scanning and analyzed with
SCION IMAGER. Results are depicted relative to mock-transfected control. Each bar rep-
resents the mean ± SE of triplicate determination of an individual experiment. The significance of the differences as compared to mock-

protein core of tyrosine phosphorylated SD-4 after
digestion of the GAG chains with heparitinases I and
III and chondroitinase ABC. The 50–55 kDa proteins
which were revealed with anti-SD-4 mAb 5G9 and
with anti-Ptyr mAb 4G10 in the respective glycosami-
nidases-treated anti-Ptyr IP and anti-SD-4 IP probably
represent dimers of tyrosine-phosphorylated SD-4.
Similar apparent relative molecular masses of the SD-4
protein core were observed in the enriched PGs from
glycosaminidases-treated cell lysates.
We then observed firstly an increase in SD-4 tyro-
sine phosphorylation, and secondly that homo- or
hetero-oligomerization of CXCR4, induced by SDF-1
on HeLa cells, was prevented if the cells were pre-
treated with heparitinases I and III. This indicates the
involvement of HS in these two events.
In this study, in parallel experiments, either the cells
were treated with heparitinases I and III or the IPs
were treated with three glycosaminidases, heparitinases
I and III and chondroitinase ABC. To preserve cell
viability, lower concentrations of heparitinases were
used to treat the cells than the IPs. According to these
different conditions, GAGs, especially chondroitin sul-
fates, were still present on SD-4, if the enzyme treat-
ment was performed on the cells. This explains why
incomplete deglycanation of SD-4 was observed if the
cells were treated with heparitinases.
Finally, we asked whether HS and SD-4 were
involved in other SDF-1-induced cellular activation
signals. As SDF-1 binding to CXCR4 activates

specific for the ectodomain of SD-1 of human origin), anti-
(SD-4) mAb 5G9 (mouse IgG2a; clone 5G9; specific for the
ectodomain of SD-4 of human origin); anti-(SD-2) (goat
IgG; specific for the C-terminal domain of syndecan-2 of
human origin) (all from Santa Cruz Biotechnology Inc,
Santa Cruz, CA, USA) or anti-(beta-glycan) Igs (goat IgG;
R & D systems, Abingdon, UK), anti CD44 mAb (mouse
IgG2a; Serotec, Oxford, UK), anti-CXCR4 mAb 12G5
(mouse IgG2a; specific for the second extracellular domain
of CXCR4; BD Bioscience Pharmingen, San Diego, USA),
or their isotypes (mouse IgG1, IgG2a or goat IgG, Jackson
Immunoresearch, Laboratories Inc. (Baltimore, MD, USA)
or BD Bioscience Pharmingen (San Diego, CA, USA), all
at 10 lg Æ mL
)1
.
Preparation of PGs
The PGs from HeLa cells lysates were enriched by anion
exchange chromatographies, as described previously [38].
Binding of biotinylated SDF-1 to electroblotted
PGs
Enriched PGs were loaded onto 12% SDS ⁄ polyacrylamide
gels (Invitrogen Corp.) under non reducing conditions
and blotted onto polyvinylidene difluoride membranes
N. Charnaux et al. Syndecan-4 is an auxiliary receptor for SDF-1/CXCL12
FEBS Journal 272 (2005) 1937–1951 ª 2005 FEBS 1947
(Amersham Pharmacia Biotech., Little Chalfont, Bucks,
UK) as described [39].
After blocking, strips were incubated for 1 h at room
temperature with biotinylated SDF-1a (6.25 nm; synthes-

droitin ABC lyase; EC 4.2.2.4) (all from Sigma–Aldrich) as
described previously [39].
Immunofluorescence staining and confocal
microscopic analysis of the cells
To determine whether SDF-1 colocalizes with SD-4, HeLa
cells were incubated with anti-(SD-4) mAb 5G9, which was
revealed by Cy-3 donkey anti-mouse Igs (1 : 400; Jackson
Immunoresearch, West Grove, PA, USA). Cells were then
subsequently incubated for 1 h at 4 °C with 1-biotinylated
SDF-1a (10 lgÆmL
)1
). Cells were then labeled for 30 min at
4 °C with a streptavidin-Alexa Fluor 488 complex (1 : 100,
Molecular Probe, Inc., Eugene, OR, USA) and fixed with
paraformaldehyde (Sigma-Aldrich). As controls, cells were
incubated with the isotypes or biotinylated SDF-1a was
omitted. Cells were mounted and observed using a Zeiss
microscope (Axiovert 135 m; Carl Zeiss AG, Go
¨
ttingen,
Germany) in conjunction with a confocal laser scanning
unit (Zeiss LSM 410).
Immunoelectron microscopy
The HeLa cells were grown until 80% confluence in multi-
well chambers. After washes with phosphate buffered saline
(NaCl ⁄ P
i
), cells were incubated for 1 h at 4 °C with anti-
(SD-4) mAb 5G9 (20 lgÆmL
)1

) mixture. It was verified that these
enzymes treatment had no effect on cell viability, as
assessed by Trypan blue exclusion dye. After washing the
cells with NaCl ⁄ P
i
supplemented with orthovanadate
(1 mm, Sigma-Aldrich), whole-cell extracts were prepared
by lysis of the cells in 20 mm Tris, 150 mm NaCl, 1 mm
orthovanadate, 1% (v ⁄ v) NP-40, 10 mm phenylmethylsulfo-
nyl fluoride, 5 mm iodoacetate, 25 mm phenanthrolin and
20 lgÆmL
)1
aprotinin (all from Sigma-Aldrich), The protein
concentration in whole-cell extracts was determined by the
BCA protein assay (Pierce). These extracts were then sup-
plemented with 10 mm dithiothreitol (Sigma-Aldrich).
Thereafter, equal amounts of proteins from these extracts
were incubated for 18 h at 4 °C with 100 lL of Protein G-
Sepharose beads (Amersham Pharmacia Biotech), precoated
either by anti-Ptyr mAb 4G10 (mouse IgG2b; Upstate Bio-
technology, Inc, Lake Placid, NY, USA), anti-SD-4 mAb
5G9, or anti-CXCR4 Abs G19 (goat IgG; specific for the
first extracellular domain of CXCR4; Santa Cruz Biotech-
nology) (each at 2 lg), as described previously [33,40,41].
To release bound components, beads were then boiled for
10 min with 300 lLof2· sample buffer for SDS ⁄ PAGE
and centrifuged (400 g; 5 min at 15 °C). Cell lysates, eluates
or eluted proteins were submitted to 12% SDS ⁄ PAGE
under non reducing conditions and then transferred onto
polyvinylidene difluoride membranes. Complexes were

kinases by SDF-1
HeLa cells were washed with NaCl ⁄ P
i
and cultured for
48 h in DMEM supplemented with 0.1% (v ⁄ v) FBS. In
some experiments, cells were pretreated for 2 h at 37 °C
with heparitinases I and III mixture, as just described. It
was verified that these enzymes treatment had no effect on
cell viability, as assessed by Trypan blue exclusion dye.
Thereafter, cells were incubated for 0–30 min at 37 °C with
SDF-1a (at 0–125 nm). After washing with NaCl ⁄ P
i
-ortho-
vanadate (1 mm), whole cell extracts were prepared [39].
The amount of protein of these extracts was controlled by
using a protein detection kit (Pierce). Equal amounts of
total proteins from these extracts were then submitted
to 10% SDS ⁄ PAGE and transferred to nitrocellulose
membrane (Amersham Pharmacia Biotech). MAPKs were
detected using polyclonal Abs, respectively, specific for
phospho-p44 ⁄ p42 [Thr202 ⁄ Tyr204], phospho-SAPK ⁄ JNK
[Thr183 ⁄ Tyr185], total p44 ⁄ p42 or total SAPK-JNK (rabbit
IgG; all from Cell Signaling Technology). Revelation was
performed as described [39]. Quantification of p44 ⁄ p42
MAPKs- and of SAPK ⁄ JNK phosphorylations was per-
formed by using the scion imager after autoradiography
scanning. For each sample, data were expressed as a ratio
of p44 ⁄ p42 MAPKs- or SAPK ⁄ JNK-phosphorylated pro-
teins over total proteins, in absorbance units. The
mean ± SE of triplicate determinations of individuals

SD-1 and CXCR4. In parallel, the protein expressions of
SD-4, SD-1, SD-2, beta-glycan, CXCR4 were analyzed by
indirect immunofluorescence and FACS analysis. SD-4
mRNA, SD-1 mRNA, SD-2 mRNA and CXCR4 mRNA
and, to normalize for input of total RNA, glyceraldehyde
3-phosphodehydrogenase (GAPDH) mRNA were quanti-
fied by RT-PCR. Total cellular RNA was extracted, using
a Qiagen RNA ⁄ DNA Mini Kit (Qiagen S.A., Cortaboeuf,
France). For this purpose, confluent monolayers of mock-
transfected HeLa cells, mutSD-4 dsRNA-transfected HeLa
cells and from SD-4 dsRNA transfected HeLa cells were
previously grown in a six-well tissue culture. Reverse tran-
scription was performed using a Advantage RT-for-PCR
Kit (BD Biosciences Clontech, Le Pont-de-Claix, France).
The following synthetic SD-4 primers were used: – upper
primer CGA GAG ACT GAG GTC ATC GAC; lower pri-
mer: CGC GTA GAA CTC ATT GGT GG. These primers
were designed to amplify a 531 bp coding sequence of SD-
4. The following SD-1 primers were used: sense primer,
5¢-TCTGACAACTTCTCCGGCTC-3¢; antisense primer:
5¢-CCACTTCTGGCAGGACTACA-3¢; these primers were
designed to amplify a 211 bp coding sequence of SD-1. The
following synthetic SD-2 primers were used: sense primer
5¢-GGGAGCTGATGAGGATGTAG-3¢; antisense primer
5¢-CACTGGATGGTTTGCGTTCT-3¢. These primers were
designed to amplify a 394 bp coding sequence of SD-2. The
following synthetic CXCR4 primers were used: sense pri-
mer: 5¢-ATCTTTGCCAACGTCAGT-3¢; antisense primer:
5¢-TCACACCCTTGCTTGATG-3¢. These primers were
designed to amplify a 308 bp coding sequence of CXCR-4.

Recherche et des Enseignements Doctoraux (Ministe
`
re
de l’Enseignement Superieur et de la Recherche), Uni-
versite
´
Paris XIII. We thank R. Fagard for his techni-
cal advices. We are grateful to J. Vaysse for her
suggestions.
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