Alpha 1,3-fucosyltransferase-VII regulates the signaling
molecules of the insulin receptor pathway
Qiu-yan Wang
1
, Ying Zhang
1
, Hai-jiao Chen
2
, Zong-hou Shen
1
and Hui-li Chen
1
1 Key Laboratory of Glycoconjugate Research, Shanghai Medical College, Fudan University, Shanghai, China
2 Department of Urology, Zhong-shan Hospital, Fudan University, Shanghai, China
Glycosylation is important and the most common form
of post-translational modification that regulates many
aspects of protein function [1,2]. In recent years,
increased attention has been paid to the relationship
between structural changes in surface glycans and sur-
face receptor signaling. It has been reported [3] that
overexpression of N-acetylglucosaminyltransferase
(GnT)-III introducing a bisecting N-acetylglucosamine
Keywords
epidermal growth factor receptor; a1,3-
fucosyltransferase-VII; human
hepatocarcinoma cell line; insulin receptor;
signaling molecules
Correspondence
H. Chen, Key Laboratory of Glycoconjugate
Research, Ministry of Health, Department of
Biochemistry, Shanghai Medical College,

expression in cells. In addition, the phosphorylation intensity and differ-
ence in phosphorylation intensity between cells with different levels of
a1,3-FucT-VII expression were attenuated significantly by the inhibitor of
InR tyrosine kinase and by the mAb to SLe
x
. Furthermore, insulin-induced
signaling was facilitated in a1,3-FucT-VII-transfected cells, particularly
FucTVII-H. These findings provide strong evidence that a1,3-FucT-VII
may affect insulin signaling by upregulating the phosphorylation and
expression of some signaling molecules involved in the InR-signaling path-
way. These effects are likely mediated by its product, SLe
x
, on the glycans
of the InR. This is the first study to report that changes in the terminal
structure of glycans on a surface receptor can modify cell signaling.
Abbreviations
CDK, cyclin-dependent kinase; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; FucT, fucosyltransferase; GlcNAc,
N-acetylglucosamine; GnT, N-acetylglucosaminyltransferase; InR, insulin receptor; IRS-1, insulin receptor substrate-1; MAPK, mitogen-
activated protein kinase; MEK, MAPK kinase; NGF, nerve growth factor; PDK-1, phosphoinositide-dependent kinase-1; PKB, protein kinase B
(Akt); PKN, novel protein kinase; TGF, transforming growth factor.
526 FEBS Journal 274 (2007) 526–538 ª 2006 The Authors Journal compilation ª 2006 FEBS
(GlcNAc) into the N-glycans of epidermal growth fac-
tor receptor (EGFR) in U373 MG glioma cells led to
decreased epidermal growth factor (EGF) binding and
autophosphorylation of EGFR, as well as reduced cell
proliferation upon EGF stimulation. It has also been
reported [4] that overexpression of GnT-III in pheo-
chromocytoma PC12 cells inhibited transient tyrosine
phosphorylation and dimerization of the nerve growth
factor (NGF) receptor (Trk) upon stimulation with

transfection of GnT-V into human fibrosarcoma
HT1080 and mouse NIH 3T3 cells to increase the Glc-
NAcb1,6-branch on N-cadherin inhibited signaling
between N-cadherin and ERK1 ⁄ 2, and consequently
reduced calcium-dependent cell–cell adhesion mediated
by N-cadherin. These results provide evidence that the
N-cadherin signaling pathway is also influenced by the
glycan structures on N-cadherin. Wang et al. [7] repor-
ted that in embryonic fibroblast cells deprived of a1,6-
fucosyltransferase (FucT-VIII), an enzyme responsible
for the synthesis of core fucose on N-glycans, EGF-
induced phosphorylation of EGFR and EGFR-
mediated JNK or ERK activation were suppressed.
Taniguchi [8] also discovered that signal transduction
of the transforming growth factor b1 (TGF b1) recep-
tor was deficient in FucT-VIII knockout mice, leading
to emphysema-like phenotypes in the lung. These
results show that the core fucose on N-glycans is essen-
tial for EGF and TGF b1 signaling. However, all the
above-mentioned structural changes in receptor glycans
are located in the core portion of N-glycan, and whe-
ther alteration of the terminal residue on the outer
chain of either N-orO-glycan can also modify surface
receptor signaling remains unclear.
It has been documented that sialyl Lewis antigens
(SLe) expressed on the surface of cancer cells, such as
SLe
x
[SAa2,3 Galb1,4 (Fuca1,3) GlcNAc-] and SLe
a

was increased when H7721 cells
were treated with proliferative inducers, and decreased
after treatment with differentiative inducers [18]. The
change in SLe
x
level was proportional to a1,3-FucT-
VII expression. Moreover, the ex vivo metastatic
potential was positively correlated with surface SLe
x
and cellular a1,3-FucT-VII levels, and could be inhib-
ited by a mAb (KM93) against surface SLe
x
[15,18].
Further studies have shown that insulin also enhanced
expression of SLe
x
and a1,3-FucT-VII and the meta-
static potential of H7721 cells [19]. In addition, our
group recently found that expression of cyclin-depend-
ent kinase (CDK) inhibitor, p27
Kip1
protein, was
decreased in H7721 cells transfected with a1,3-FucT-
VII cDNA. Uninhibited CDK2 resulted from a reduc-
tion in the p27
Kip1
-stimulated phosphorylation of
retinoblastoma protein, facilitating G
1
⁄ S transition and

receptor substrate-1 (IRS-1), PKB, phosphoinositide-
dependent kinase-1 (PDK-1), novel protein kinase
(PKN), p42 ⁄ 44 MAPK and MEK were analyzed as
the signaling molecules involved in InR signaling
[19,23]. Expression of b catenin and its downstream
transcription factor TCF in the Wnt signaling pathway
[24], which cross-talks with the InR pathway was
also studied. Mock cells transfected with the vector
pcDNA3.1 were used as controls.
Results
Characterization of two a1,3-FucT-VII-transfected
cell lines
As shown in Fig. 1A,B, a1,3-FucT-VII mRNA was
increased significantly in H7721 cells transfected with
a1,3-FucT-VII cDNA. In FucTVII-M (moderate
expression) and FucTVII-H (high expression) cells, it
was upregulated to 373.3 and 613.3% of the mock-
transfected cell level, respectively (both P < 0.01).
Consequently, expression of SLe
x
, the product of a1,3-
FucT-VII, was also elevated on the cell surface, to
171 and 284% of the mock-transfection value in
FucTVII-M and FucTVII-H cells, respectively (both
P < 0.01; Fig. 1C,D).
Expression of InR-a, EGFR and their SLe
x
in
a1,3-FucT-VII-transfected H7721 cells
Expression of cell-surface InR-a and EGFR were ana-

D
C
Mock
FucTVII-M
FucTVII-M FucTVII-H
FucT-VII
β
-actin
FucTVII-H
497 bp
789 bp
0
10
0
10
1
10
2
10
3
10
4
FL1-H
( - ) Control Mock
10
0
10
1
10
2

200
Counts
0 200
Counts
0 200
Counts
0 200
Counts
Fig. 1. Characterization of a1,3-FucT-VII cDNA-transfected H7721 cells. (A) RT-PCR profiles of a1,3-FucT-VII mRNA in mock- and a1,3-FucT-
VII-transfected cells. (B) Relative expressions of a1,3-FucT-VII mRNA in mock- and a1,3-FucT-VII transfected cells (n ¼ 3). (C) Fluorescence-
activated cell spectra of cell-surface SLe
x
on mock- and a1,3-FucT-VII transfected cell lines. (D) Relative expressions of surface SLe
x
on
mock- and a1,3-FucT-VII transfected cells (n ¼ 3). Mock, H7721 cells transfected with pcDNA3.1 vector; FucTVII-M, H7721 cell line with
moderate expression of transfected a1,3-FucT-VII; FucTVII-H, H7721 cell line with high expression of transfected a1,3-FucT-VII. (A) and (C)
are representative of three reproducible experiments. *P < 0.01 compared with ‘Mock’. RT-PCR and flow cytometry are described in the
Experimental procedures.
Fucosyltransferase-VII regulates insulin signaling Q. Wang et al.
528 FEBS Journal 274 (2007) 526–538 ª 2006 The Authors Journal compilation ª 2006 FEBS
results in Fig. 2A,B show that their expression was not
obviously changed in a1,3-FucT-VII-transfected cells
compared with mock-transfected cells. Results from
western immunoblots indicated that protein expression
in InR-a and EGFR was also unchanged following
transfection with a1,3-FucT-VII (Fig. 2C). However,
after immunoprecipitation and western blotting of
these receptors, and using KM93 as the probe for
SLe

Mock
FucTVII-M
FucTVII-H
AB
0
10
0
10
1
10
2
10
3
10
4
FL1-H
M1
( - ) Control-InR ( - ) Control-EGFR
FucTVII-H-InR
0
50
100
150
200
250
300
350
400
450
500

FL1-H
M1
200
Counts
0
10
0
10
1
10
2
10
3
10
4
FL1-H
M1
M1
200
Counts
0
10
0
10
1
10
2
10
3
10

10
0
10
1
10
2
10
3
10
4
FL1-H
M1
200
Counts
0
200
Counts
10
0
10
1
10
2
10
3
10
4
FL1-H
M1
M1

Experimental procedures.
Q. Wang et al. Fucosyltransferase-VII regulates insulin signaling
FEBS Journal 274 (2007) 526–538 ª 2006 The Authors Journal compilation ª 2006 FEBS 529
(both P < 0.01), whereas that of EGFR was
unchanged.
Tyrosine phosphorylation of IRS-1 occurs earlier in
insulin signaling. The IRS-1 protein was decreased in
a1,3-FucT-VII-transfected cells, although the change
was not statistically significant. When relative tyrosine
phosphorylation was calculated as above, it was found
that the level of phosphorylated IRS-1 was increased
to 2.8 and 8.5 times that of the mock-transfection
value in FucTVII-M and FucTVII-H cells, respectively
(both P < 0.01; Fig. 3C,D).
Phosphorylation and activity of PKB, expression
of PDK-1, PKN and phospho-PKN in
a1,3-FucT-VII-transfected H7721 cells
In insulin signaling, activation of PKB has been impli-
cated as a key step and it also has a major role in the
physiological effects of insulin [25]. As shown in
Fig. 4A,B, expression of PKB protein was not obvi-
ously altered in a1,3-FucT-VII-transfected H7721 cells,
but relative phosphorylation at T308 and S473 in PKB
(calculated from the ratio of the staining intensity of
phosphorylated protein to unphosphorylated protein
after normalization with b-actin) was apparently eleva-
ted when compared with mock-transfected cells. After
densitometric quantification, relative T308 phosphory-
lation was 149 and 205% of the mock-transfection
level in FucTVII-M and FucTVII-H cells, respectively

200
250
300
350
400
450
Relative intensity
kcoM
M
-
II
V
Tc
u
F
H
-IIVT
c
u
F
InR-β InR-β-Tyr-p
EGFR-Tyr-pEGFR
D
0
100
200
300
400
500
600

WB, western immunoblot with the antibody to phosphotyrosine (PT66) or to the protein indicated on the right; IRS-1, insulin receptor sub-
strate-1; Tyr-p, tyrosine phosphorylated. (A) and (C) are representative of three reproducible experiments. *P < 0.01 compared with ‘Mock’.
Immunoprecipitation and western immunoblotting are described in the Experimental procedures.
Fucosyltransferase-VII regulates insulin signaling Q. Wang et al.
530 FEBS Journal 274 (2007) 526–538 ª 2006 The Authors Journal compilation ª 2006 FEBS
PDK-1 ⁄ PKB pathway in the insulin receptor [23]. Fig-
ure 5A,B shows that expression of MEK and p42 ⁄ 44
MAPK proteins was not apparently altered in a1,
3-FucT-VII-transfected H7721 cells. However, expres-
sion of c-Raf-1 increased significantly following a1,3-
FucT-VII transfection, being 168 and 325% of the
mock-transfection value in FucTVII-M and FucTVII-
H cells, respectively (both P < 0.01). Moreover, the
relative phosphorylation of MEK, as determined by
the ratio of p-MEK to MEK, was upregulated to 207
and 425% in FucTVII-M and FucTVII-H cells, respec-
tively (both P < 0.01), and the relative phosphoryla-
tion of p42 ⁄ 44 MAPK (the ratio of p-p42 ⁄ 44 MAPK
to p42⁄ 44 MAPK) was also increased in FucTVII-M
and FucTVII-H cells, being 2.82 and 6.01 times the
mock-transfection value (both P < 0.01).
Effect of HNMPA-(AM)
3
and KM93 on the
phosphorylation of PKB and p42/44 MAPK
In order to study whether the alteration in the phos-
phorylation of PKB and p42 ⁄ 44 MAPK was mediated
by InR kinase and surface SLe
x
, phosphorylation of

phorylation intensities among the three cell lines were
also decreased significantly (Fig. 6C,D). The reduction
in phosphorylation of PKB and p42 ⁄ 44 MAPK in
a1,3-FucT-VII-transfected cells was  41.1–89.7%
(P<0.01). In the presence of HNMPA-(AM)
3
or
KM93, the rate of inhibition of phosphorylation was
correlated with expression of a1,3-FucT-VII, which was
FucTVII-H > FucTVII-H > mock-transfected cells.
However, some differences in the phosphorylation
intensities of PKB and MAPK were observed in mock-
and a1,3-FucT-VII-transfected cells in the presence of
both inhibitors, but the differences were either not sta-
tistically significant or P < 0.05.
AC
D
E
F
B
0
05
001
0
5
1
002
052
003
053

ti
sn
e
tni
e
vi
t
aleR
Mock
FucT-VII-M
FucT-VII-H
Mock
PKB-T308
PKB-S473
PKB
β-actin
FucTVII-M FucTVII-H
Mock
Phosphorylated
GSK3 α/β
β-actin
FucTVII-M FucTVII-H
Mock
PDK-1
PKN
p-PKN
β-actin
FucTVII-M FucTVII-H
Fig. 4. Effects of a1,3-FucT-VII transfection on the phosphorylation, protein expression and activity of some signaling molecules. (A) Western
blot profiles of PKB and T308-, S473-phosphorylated PKB after staining with specific antibodies and horseradish peroxidase-labeled sec-

found that phospho-PKB-S473 was barely seen in
insulin-untreated and serum-starved cells, but was
expressed in insulin-treated cells. By contrast, phospho-
PKB-T308 and phospho-p42 ⁄ 44 MAPK were expres-
sed in both insulin-untreated and insulin-treated cells.
The intensity levels for phospho-PKB-T308 and phos-
pho-MAPK in both insulin-untreated and -treated
cells, as well as the phospho-PKB-S473 in insulin-trea-
ted cells, were FucTVII-H > FucTVII-M > mock
(Fig. 8A). In the presence of insulin, phosphorylation
of PKB and MAPK was obviously upregulated, and
was significantly higher than in the corresponding
control cells cultured in the absence of insulin. The
response to insulin stimulation was proportional to the
expression of a1,3-FucT-VII. In insulin-stimulated
FucTVII-M and FucTVII-H cells, phospho-PKB-T308
was upregulated to 215 and 398% of the mock-transfc-
tion level (both P < 0.01), and phospho-PKB-S473
was upregulated to 192 and 354% of the mock-trans-
fection level, respectively (both P < 0.01). Similarly,
phospho-MAPK was 184 and 345% of the mock-
transfection level, respectively (both P < 0.01)
(Fig. 8B).
Discussion
The results shown in Fig. 1A,B indicate that two a1,3-
FucT-VII-transfected cell lines were established with
moderate and high expression of the exogenous
cDNA. Expression of SLe
x
, the product of a1,3-FucT-

08
4
4
/24
p
KE
M
fa
R-c
KP
A
M
44/
24
p-
p
KEM-
p
KP
AM
Relative intensity
k
co
M
B
M-I
IV
TcuF
H-IIVTcuF
Mock

not suitable substrates for fucosylation by exogenous
a1,3-FucT-VII. In other words, SLe
x
on EGFR is
probably not synthesized by a1,3-FucT-VII, but by
other a1,3-FucTs.
Our findings showed that transfection of a1,3-FucT-
VII promoted the functional activity of InR, as verified
by increased tyrosine phosphorylation of InR-b and
IRS-1 (Fig. 3). Moreover, Ser ⁄ Thr phosphorylation of
InR signaling molecules, including PKB (Fig. 4A,B),
MEK, p42 ⁄ 44 MAPK (Fig. 5A,B) and the activity of
PKB (Fig. 4C,D) was stimulated concomitantly.
Expression of some other signaling proteins, such as
PDK-1, PKN (Fig. 4E,F), c-Raf-1 (Fig. 5A,B) and
b-catenin (Fig. 7A,B), was also upregulated by a1,3-
FucT-VII. Elevation of Ser ⁄ Thr phosphorylation in
downstream signaling molecules was presumed to be
mediated by increased tyrosine phosphorylation of InR
and IRS-1; the latter resulting from the increased SLe
x
content of InR-a. This speculation was evidenced by
the following. First, the intensity of Ser ⁄ Thr phos-
phorylation in downstream signaling molecules was
positively correlated with the intensity of tyrosine
phosphorylation in InR and IRS-1, and tyrosine phos-
phorylation was proportional to the SLe
x
content of
InR-a and also the mRNA expression of a-1,3-FucT-

2
003
004
005
006
700
3)MA
(
-AP
M
N
H
ht
iW
3
)M
A(-
A
PMNHtuoht
i
W
Relative intensity
803T-BKP
374S-BKP
KPAM44/24p-p
*
*
*
*
#

KP
p374S-BK
P
KPAM44/24p-p
*
*
*
*
*
*
#
#
#
#
Mock-1
AB
C
D
FucTVII-M
( ) HNMPA-(AM)3
PKB-T308p
PKB-S473p
p-p42/44 MAPK
β-actin
(+) HNMPA-(AM)3
FucTVII-H
Mock-1
FucTVII-M
FucTVII-H
Mock-2

FucT-VII; FucTVII-H, H7721 cell line with high expression of transfected a1,3-FucT-VII; PKB-T308p, phosphorylated PKB at T308; PKB-S473p,
phosphorylated PKB at S473; p-p42 ⁄ 44 MAPK, phosphorylated p42 ⁄ 44 MAPK; Mock-1, mock cells without HNMPA-(AM)
3
or KM93 treat-
ment; Mock-2, mock cells with HNMPA-(AM)
3
or KM93 treatment. (A) and (C) are representative of three reproducible experiments.
*P < 0.01 compared with ‘Mock-1’. #P < 0.05 compared with ‘Mock-2. Western immunoblotting is described in the Experimental proce-
dures. Samples without and with HNMPA-(AM)
3
or KM93 were examined simultaneously on the same electrophoresis gel.
Q. Wang et al. Fucosyltransferase-VII regulates insulin signaling
FEBS Journal 274 (2007) 526–538 ª 2006 The Authors Journal compilation ª 2006 FEBS 533
appears that upregulation of phosphorylation and pro-
tein expression was not mediated by EGFR, because
the SLe
x
content and tyrosine autophosphorylation of
EGFR remained constant following a1,3-FucT-VII
transfection.
In a previous insulin stimulation experiment, it was
found that H7721 cells were prone to die in serum-
free (0%) medium; therefore, 2% fetal bovine serum-
deficient medium was used. The results showed that
phospho-PKB-S473 barely appeared in cells cultured
in the serum-deficient medium (Fig. 8A). As shown
in Fig. 4A, however, there was basal expression of
both phospho-PKB-T308 and phospho-PKB-S473 in
mock- and a1,3-FucT-VII-transfected cells. These
observations suggest that phospho-PKB-T308 and

coM
M-7T
F
H-7
T
F
*
*
*
*
*
*
Mock
A
B
FT7M
( ) Insulin (+) Insulin
FT7H
Mock
FT7M
FT7H
PKB-T308p
PKB-S473p
p-p42/44 MAPK
β-actin
Fig. 8. Facilitation of insulin signaling in a1,3-FucT-VII-transfected
cells. (A) Western profiles of phosphorylated PKB and p22 ⁄ 24
MAPK in insulin-untreated and -treated cells cultured in 2% fetal
bovine serum medium. (B) Quantification of phosphorylated PKB
and p22 ⁄ 24 MAPK in the presence of insulin (n ¼ 3). Mock, H7721

*
*
Mock FucTVII-M FucTVII-H
Mock FucTVII-M FucTVII-H
Mock FucTVII-M FucTVII-H
β-actin
β-Catenin
Relative intensity
Fig. 7. Effects of a1,3-FucT-VII cDNA transfection on the expres-
sion of b-catenin and TCF activity. (A) Western blot profile of
b-catenin. (B) Densitometric quantification of (A) (n ¼ 3). (C) Trans-
activation activity of TCF measured as luciferase activity (n ¼ 3).
Mock, H7721 cells transfected with pcDNA3.1 vector; FucTVII-M,
H7721 cell line with moderate expression of transfected a1,3-FucT-
VII; FucTVII-H, H7721 cell line with high expression of transfected
a1,3-FucT-VII; TCF, T-cell factor (transcription factor). (A) is repre-
sentative of three reproducible experiments. *P < 0.01 compared
with ‘Mock’. Western blotting and luciferase assay are described in
the Experimental procedures.
Fucosyltransferase-VII regulates insulin signaling Q. Wang et al.
534 FEBS Journal 274 (2007) 526–538 ª 2006 The Authors Journal compilation ª 2006 FEBS
because a reduction in rictor or mammalian TOR
(mTOR) expression inhibited the signaling of PKB.
Rictor–mTOR complex can also facilitate the phos-
phorylation of PKB-T308 by PDK-1 [33]. Basal
expression of phospho-PKB-S473 in serum-containing
medium may result from the stimulation of insulin or
growth factors in the 10% fetal bovine serum. Our
finding that the cell response to insulin was correla-
ted with expression of a1,3-FucT-VII and SLe

a1,3-FucT-VII expression after transfection of anti-
sense a1,3-FucT-VII cDNA was not apparent,
because parent H7721 cells express a low level of
endogenous a-1,3-FucT-VII. Sometimes antisense
cDNA even led to cell death. When a gene of a1,3-
FucT-VII was knocked-out, almost all cells died
within 24 h. This suggests a1,3FucT-VII is essential
for the survival of H7721 cells. Therefore, construc-
tion of a plasmid containing a mutant at the cata-
lytic domain of a1,3-FucT-VII with deletion of
enzyme activity of its coding protein is very critical
if we are to determine whether the changed phospho-
rylation of signaling molecules was mediated by the
altered amount of SLe
x
on InR. This is being inves-
tigated in our laboratory.
It would be of interest to study whether the SLe
x
level of InR on insulin-responsive cells in diabetic
patients was changed. This may reveal the role of the
sugar chains on InR in the pathogenesis of diabetes.
In summary, the cDNA of a1,3-FucT-VII is able to
regulate the phosphorylation and expression of some
signaling molecules in the InR pathway, and these
effects of a1,3-FucT-VII are probably mediated by its
product, SLe
x
, on the glycans of cell-surface receptors.
Increased expression and phosphorylation of insulin-

EGFR mAb CF4 (against intracellular domain), fluorescein
isothiocyanate-conjugated and horseradish peroxidase-labe-
led secondary antibodies (goat anti-mouse and anti-rabbit
IgG) were from Sigma (St. Luois, MO). Trizol, AMV
reverse transcriptase, transcription factor TCF analysis kit
(Dual-luciferase
R
reporter assay system) and Renilla lucif-
erase reporter plasmid (pRL-TK) were from Promega
(Madison, WI). The TCF reporter plasmid (TK-luciferase
reporter) was the product of Upstate Biotechnology (Lake
Placid, NY). The RT-PCR primer of a1,3-FucT-VII was
provided by TaKaRa Co. (Tokyo, Japan). Other reagents
were commercially available in China.
a1,3-FucT-VII transfected human hepatocarcinoma
H7721 cell lines were established as previously reported
[15].
Q. Wang et al. Fucosyltransferase-VII regulates insulin signaling
FEBS Journal 274 (2007) 526–538 ª 2006 The Authors Journal compilation ª 2006 FEBS 535
Cell culture and treatment
Cells were cultured at 37 °C, 5%CO
2
in RPMI-1640 con-
taining 10% fetal bovine serum, penicillin and streptomy-
cin as described previously [5,20]. In the treatment of InR
kinase inhibitor and SLe
x
mAb, the final concentration of
HNMPA-(AM)
3

were obtained by the intensity ratios of a1,3FucT-VII ⁄
b-actin band.
Detection of the expression of SLe
x
, InR-a
subunit and EGFR on the cell surface using flow
cytometry
After being washed with NaCl ⁄ P
i
and blocked with 1%
BSA, the EDTA-detected cells (1 · 10
6)
were incubated
with 1 : 50 SLe
x
antibody KM93, 2.5 lgÆmL
)1
polyclonal
antibody against InR-a subunit or 528 mAb against the
EGFR extracellular domain for 45 min at 4 °C. In the
‘(–) Control’ sample the primary antibody was omitted.
Washed cells were incubated with 1 : 128 fluorescein
isothiocyanate-conjugated secondary antibody for 30 min
at 4 °C. cells were then suspended in NaCl ⁄ P
i
and sub-
jected to flow cytometry. Fluorescence-activated cell spec-
tra were drawn automatically, and the relative amount of
surface SLe
x

Determination of tyrosine phosphorylation
of InR-b, EGFR or IRS-1 using western
immunoblotting
Monolayer cells were lysed with 200 lL lysis buffer as
described above. Immunoprecipitated InR-b or EGFR was
divided into two (for different probes) and subjected to
8% SDS ⁄ PAGE and western blotting, the membranes
were probed with 1 : 1000 phosphotyrosine mAb (PT66)
and InR-b antibody or 1 : 500 EGFR antibody (CF4) in
Tris-buffered saline with 5% fat-free dry milk, followed
by incubation with 1 : 500 diluted horseradish peroxidase-
labeled secondary antibody. The color was also developed
with an enhanced chemiluminescence reagent. The meas-
urement of tyrosine phosphorylation of IRS-1 was similar
to that of InR-b and EGFR, but the primary antibodies
used in western immunoblotting were anti-PT66 and anti-
IRS-1 sera.
Analysis of the proteins or phosphorylated
proteins of PKB, PDK-1, PKN, Raf-1, MEK, p42/44
MAPK and b-catenin using western
immunoblotting
Briefly, cells were homogenized in Mes buffer (0.1 m
pH 6.5, 150 mm NaCl, 2% Triton X-100, 25% glycerol,
1mm phenylmethylsulfonyl fluoride, 1 mg % leupeptin
Fucosyltransferase-VII regulates insulin signaling Q. Wang et al.
536 FEBS Journal 274 (2007) 526–538 ª 2006 The Authors Journal compilation ª 2006 FEBS
and pepstatin), and 50 lg supernatant protein after cen-
trifugation were subjected to 10% SDS ⁄ PAGE. The mem-
branes were treated with one of the 1 : 500-diluted
primary antibodies of the determined proteins or phos-

gents [5].
Determination of TCF transcription factor activity
with dual luciferase reporter system
The method was performed according to the protocol of
‘dual-luciferase
Ò
reporter assay’ in the manual. In brief,
0.5 lg TCF reporter plasmid and 0.4 lg pRL-TK plasmid
was mixed in 25 lL antibiotic- and serum-free RPMI-1640
(ASF-RPMI), and 2 lL Lipofectamine
TM
was added to
23 lL of the above medium. Two separate preparations
were mixed within 5 min. After standing at room tempera-
ture for 30 min and adding 100 lL ASF-RPMI, the
150 lL plasmid ⁄ Lipofectamine mixture was added to the
ASF-RPMI washed cells already cultured in 12-well plate
for 24 h according the Lipofectamine
TM
manual. Cells
were further cultured at 37 °C, 5% CO
2
for 5 h, and
transferred to 300 lL antibiotic-free RPMI-1640 contain-
ing 20% serum for 24 h incubation. Finally, the cells were
incubated in normal RPMI-1640 for 48 h. After washing
with NaCl ⁄ P
i
, cells were lyzed and luciferase activity was
assayed according to the manual provided with the kit.

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