Expression and function of Noxo1c, an alternative splicing
form of the NADPH oxidase organizer 1
Ryu Takeya
1,2
, Masahiko Taura
1
, Tomoko Yamasaki
1
, Seiji Naito
3
and Hideki Sumimoto
1,2
1 Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan
2 CREST, Japan Science and Technology Agency, Saitama, Japan
3 Department of Urology, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan
Members of the NADPH oxidase (Nox) family pro-
duce superoxide from molecular oxygen in conjunction
with oxidation of NADPH [1–10]. Superoxide gener-
ated serves as a precursor of other reactive oxygen spe-
cies, which are currently considered to be involved
in various physiological processes. The founder Nox
enzyme gp91
phox
, also termed Nox2, is predominantly
expressed in professional phagocytes, and plays a cru-
cial role in host defense; superoxide generation by
gp91
phox
leads to subsequent formation of microbicidal
reactive oxygen species such as hydroxyl radical and
hypochlorous acid. Nox1, the second member of the

2006)
doi:10.1111/j.1742-4658.2006.05371.x
Activation of the superoxide-producing NADPH oxidase Nox1 requires
both the organizer protein Noxo1 and the activator protein Noxa1. Here
we describe an alternative splicing form of Noxo1, Noxo1c, which is
expressed in the testis and fetal brain. The Noxo1c protein contains an
additional five amino acids in the N-terminal PX domain, a phosphoinosi-
tide-binding module; the domain plays an essential role in supporting
superoxide production by NADPH oxidase (Nox) family oxidases including
Nox1, gp91
phox
⁄ Nox2, and Nox3, as shown in this study. The PX domain
isolated from Noxo1c shows a lower affinity for phosphoinositides than
that from the classical splicing form Noxo1b. Consistent with this, in rest-
ing cells, Noxo1c is poorly localized to the membrane, and thus less effect-
ive in activating Nox1 than Noxo1b, which is constitutively present at the
membrane. On the other hand, cell stimulation with phorbol 12-myristate
13-acetate (PMA), an activator of Nox1–3, facilitates membrane transloca-
tion of Noxo1c; as a result, Noxo1c is equivalent to Noxo1b in Nox1 acti-
vation in PMA-stimulated cells. The effect of the five-amino-acid insertion
in the Noxo1 PX domain appears to depend on the type of Nox; in activa-
tion of gp91
phox
⁄ Nox2, Noxo1c is less active than Noxo1b even in the
presence of PMA, whereas Noxo1c and Noxo1b support the superoxide-
producing activity of Nox3 to the same extent in a manner independent of
cell stimulation.
Abbreviations
CHO, Chinese hamster ovary; GST, glutathione S-transferase; HA, hemaglutinin; Nox, NADPH oxidase; Noxo1, Nox organizer 1; Noxa1,
Nox activator 1; PMA, phorbol 12-myristate 13-acetate; PtdIns(3)P, phosphatidylinositol 3-phosphate; PtdIns(4)P, phosphatidylinositol

tion. On the other hand, Noxo1 lacks the
autoinhibitory region [15,17,18]; its SH3 domains are
capable of binding to p22
phox
even in a resting state
[15]. This seems to explain why cell stimulation with
phorbol 12-myristate 13-acetate (PMA), a potent acti-
vator of protein kinase C, is not required for Noxo1-
dependent superoxide-producing activity of Nox1 [15].
In addition to the SH3 domains, Noxo1 and p47
phox
harbor a phagocyte oxidase (phox) homology (PX)
domain in the N-terminus. PX domains occur in a
variety of proteins involved in cell signaling, mem-
brane trafficking, and polarity establishment, and func-
tion as phosphoinositide-binding modules in the
assembly of proteins at membrane surfaces [20–22].
Through the interaction with phosphoinositides, the
PX domain of p47
phox
plays a crucial role in mem-
brane recruitment of the protein and subsequent
activation of the phagocyte oxidase [23]. The phospho-
inositide-binding activity of the Noxo1 PX domain
seems also to be involved in activation of Nox1 [19].
The third oxidase, Nox3, is involved in otoconia forma-
tion in mouse inner ears [24], and appears to be consti-
tutively active even in the absence of an oxidase
organizer (p47
phox

script possesses the activity to support activation of
the Nox enzymes. In this study, we show the expres-
sion of alternatively spliced transcripts of the NOXO1
gene, by PCR using variant-specific primers, and the
roles of the protein products in activation of Nox
oxidases.
Results and Discussion
Alternative splice forms of human Noxo1
We have previously identified a transcript (AB097667)
of human NOXO1 gene encoding 371 amino acids
[15], which is identical with that reported by Ba
´
nfi
et al. (AF539796) and Cheng & Lambeth (AF532984)
[17,19] (Fig. 1A). On the other hand, Geiszt et al.
reported the alternative transcript (AY255768) [18],
which encodes a protein lacking Lys50. To investigate
the relative abundance of spliced variants of Noxo1,
we performed PCR experiments using cDNA panels as
template, and sequenced the PCR products (for details,
see Experimental procedures). The sequencing analysis
revealed that the transcript that we have previously
reported (AB097667, AF532984, and AF539796), cur-
rently referred to as Noxo1b [28], is the major mRNA
form in various human tissues including the colon.
Another alternative transcript, Noxo1c (AF532985), is
abundantly expressed in the testis. This transcript is
generated by the use of the alternative splice donor site
of the ends of exon 3 (Fig. 1A) and thus contains five
additional amino acids in the PX domain (Fig. 1B).

phosphoinositides. The other two variants with dele-
tion of Lys50, Noxo1a (AY255768 and AF532983)
and Noxo1d (AY191359), have been deposited in the
GenBank database: the deletion in these variants is
generated by alternative splicing involved in a different
splice acceptor site of exon 3. In the present PCR
experiments, Noxo1a was expressed in skeletal muscle
and Noxo1d in the brain; these two variants were
expressed to a much lesser extent than Noxo1b and
Noxo1c (data not shown).
A
C
B
Fig. 1. Alternative splice forms of human Noxo1. (A) The genomic organization of human NOXO1 gene. Translated sequences are shown as
black boxes, and untranslated sequences as open boxes. In the lower panel, sequence around splice sites of the 3rd exon are shown. Intron
sequences are shown in lower case, and exon sequences in upper case. A five-amino-acid insertion of Noxo1c is underlined. (B) Schematic
presentation of domain structures of Noxo1 and the location of the five-amino-acid insertion in the PX domain. SH3, Src-homology 3 domain;
PRR, proline-rich region. (C) Sequence alignments of the PX domains of Noxo1, p47
phox
, p40
phox
, SNX3, and Vam7p. The alignments take
the secondary structure of the p47
phox
PX domain into account [29]. A consensus sequence is shown on the top, where # indicates hydro-
phobic residues. A five-amino-acid insertion of Noxo1c is highlighted. Lys92 in p40
phox
and Lys79 in p47
phox
, mentioned in the text, are

delineate the difference in the two products, we subjec-
ted the PCR fragments to PAGE; the two fragments
were clearly separated. As shown in Fig. 2E, Noxo1c
and Noxo1b were almost equally expressed in the tes-
tis, whereas Noxo1b was the major form in the colon.
To investigate the physiological relevance of Noxo1c
expression in the testis, we examined expression of
Nox1 and Noxa1 by PCR analysis and found a small
but significant amount of the Nox1 and Noxa1
mRNAs in the testis (data not shown), which is consis-
tent with the previous observation by Cheng et al. [32].
It has also been shown that Nox1 is present in the
androgen-independent prostate cancer LNCaP cells
[33]. RT-PCR analysis revealed that LNCaP cells
abundantly expressed the mRNA for Noxo1c
(Fig. 2F). The Noxo1c mRNA also existed in several
Nox1-expressing human cancer cell lines: the andro-
gen-independent prostate cancer PC3 and DU145 cells
and the testicular germ cell tumor NEC8 cells
(Fig. 2F). Noxa1, a protein that activates Nox1 in co-
operation with Noxo1 [15,17–19], was also expressed
(Fig. 2F) in LNCaP, PC3, and DU145 cells, suggesting
that Nox1 is regulated by Noxo1c and Noxa1 in these
cancer cells. The role of Nox1 in prostate tumors has
been suggested: Nox1 seems to increase tumorigenicity
of DU145 prostate cancer cells [34]; and increased
expression of endogenous Nox1 is observed in parallel
with increasing tumor and metastatic potential in a
series of cell lines developed from LNCaP cells [35]. As
the mRNA for Noxo1c was detected in fetal brain

D
E
F G
R. Takeya et al. Expression and function of Noxo1c
FEBS Journal 273 (2006) 3663–3677 ª 2006 The Authors Journal compilation ª 2006 FEBS 3667
lobe, parietal lobe, pons, cerebellum, and spinal cord,
suggestive of the role in neurons.
Activation of Nox1 by Noxo1b and Noxo1c in
Chinese hamster ovary (CHO) cells
It is well known that the classical splicing form
Noxo1b is essential for activation of Nox1 [15,17–19].
To know the activity of Noxo1c to activate Nox1, we
transfected Chinese hamster ovary (CHO) cells with
pcDNA3.0–Nox1, pEF-BOS–p22
phox
, pEF-BOS–
Noxa1, and pEF-BOS–Noxo1b or pEF-BOS–Noxo1c.
As shown in Fig. 3A, Noxo1b and Noxo1c equally
supported Nox1 activation on stimulation with PMA.
Without stimulants added, Noxo1c also activated
Nox1 but to a lesser extent than Noxo1b (Fig. 3A,B).
On the other hand, Noxo1a, a minor spliced tran-
script, was much less active even in the presence of
PMA (Fig. 3B). We further investigated the stimulus-
independent activity of Nox1 using CHO cells trans-
fected at various amounts of the Noxo1b or Noxo1c
cDNA. As shown in Fig. 3C, Noxo1c was less active
than Noxo1b in resting cells, indicating the difference
between Noxo1b and Noxo1c.
In the above experiments, we used CHO cells

, pEF-BOS–myc-Noxa1, and simultaneously with
pEF-BOS–HA-Noxo1b, pEF-BOS–HA-Noxo1c, or pEF-BOS–HA-
Noxo1a. Superoxide production was assayed by chemilumines-
cence using Diogenes in the presence or absence of PMA
(200 ngÆmL
)1
). Each graph represents the mean ± SD of the peak
chemiluminescence values obtained from three independent trans-
fections. (C) CHO cells were transfected simultaneously with
pcDNA3.0–Nox1 (1 lg), pEF-BOS–p22
phox
(1 lg), pEF-BOS–myc-
Noxa1 (1 lg), and the indicated amount of pEF-BOS–HA-Noxo1b
(left panel) or pEF-BOS–HA-Noxo1c (right panel). Superoxide pro-
duction was assayed by chemiluminescence using Diogenes in the
presence or absence of PMA (200 ngÆmL
)1
), and expressed as the
percentage activity relative to that of 1 lg pEF-BOS–HA-Noxo1b
(left panel) or pEF-BOS–HA-Noxo1c (right panel)-transfected cells in
the presence of PMA. (D) Superoxide production in adherent CHO
cells undetached from culture dishes. CHO cells were cotransfect-
ed with pcDNA3.0–Nox1, pEF-BOS–p22
phox
, pEF-BOS–myc-Noxa1,
and simultaneously with pEF-BOS–HA-Noxo1b, pEF-BOS–HA-
Noxo1c. PMA-independent superoxide production was assayed by
chemiluminescence using Diogenes in the presence or absence of
superoxide dismutase (50 lgÆmL
)1

of Noxo1c with the membrane-integrated protein
p22
phox
, the partner of Nox1 (Fig. 4A), may be consis-
tent with the finding that Noxo1c weakly supports
superoxide production by Nox1 in resting CHO cells
more weakly than Noxo1c (Fig. 3). We also attempted
but failed to assess the intracellular localization of
Noxo1b after treatment of the cells with PMA, as the
cells became rounded without detaching from cover-
slips. To biochemically assess the localization of
Noxo1s after treatment with PMA, we prepared the
membrane fraction and tested the localization of
Noxo1c. As shown in Fig. 4B, Noxo1c localized to
the membrane only partly in resting cells, but was fur-
ther targeted to the membrane after stimulation with
PMA. On the other hand, Noxo1b was constitutively
A
C
PMA: (–) (+)
1 o x o N
β
β
1 o x o N
γ
γ
1 o x o N
β
β
1 o x o N

Noxo1 γ
γ
p22
phox
Merge
n
o i
t
a z i l
a c o l e n a r b m e m
) e
s a
e r c
n
i d
l o f (
Fig. 4. Intracellular localization of Noxo1b
and Noxo1c in CHO cells. (A) Intracellular
localization of Noxo1b (upper panels) and
Noxo1c (lower panels) in quiescent CHO
cells. In merged images (right panels), local-
ization of Noxo1b and Noxo1c is shown in
green, and p22
phox
in red. Scale bars,
20 lm. (B) Membrane translocation of
Noxo1c. Before or after cell stimulation with
PMA (200 ngÆmL
)1
), the cell lysates were

brane translocation of this protein. As Noxo1 also has
several potential protein kinase C phosphorylation
sites, Noxo1 might become phosphorylated in PMA-
stimulated cells, leading to membrane translocation.
Phosphoinositide-binding activity of the PX
domains of Noxo1b and Noxo1c
The membrane localization of Noxo1b is mediated in
part by binding of the PX domain to membrane phos-
pholipids [19]. Less association of Noxo1c with the
membrane (Fig. 3) raised the possibility that the phos-
pholipid-binding activity of Noxo1c may be impaired.
In this context, it should be noted that Noxo1c contains
the five-amino-acid insertion in the PX domain (Fig. 1).
To determine the effect of the insertion, we examined
the phosphoinositide-binding activity of the PX domain
of Noxo1c by an overlay assay, in which each phospho-
inositide was spotted on the membrane and overlaid
with glutathione S-transferase (GST)-fused PX
domains. The PX domain of Noxo1b bound to phos-
phatidylinositol 3,5-bisphosphate [PtdIns(3,5)P
2
] with
the highest affinity, which is consistent with the recent
report of Cheng & Lambeth [19]; it also interacted with
PtdIns(3)P and phosphatidylinositol 4-phosphate
[PtdIns(4)P], but to a lesser extent (Fig. 5A). On the
other hand, the PX domain of Noxo1c showed a weaker
binding activity to the phosphoinositides under the same
experimental conditions (Fig. 5B). Moreover, a lipo-
some-binding assay also showed that the Noxo1c PX

of the five-amino-acid insertion in the Noxo1 PX
domain depends on the type of Nox.
Role of the interaction between Noxo1c and
p22
phox
in Nox1-dependent and Nox3-dependent
superoxide production
It is known that Noxo1 functions via the SH3-mediated
interaction with p22
phox
, which forms a heterodimer
GST-Noxo1
β
β
-PX GST-Noxo1
γ
-PX
AB
PI
PI3P
PI4P
PI5P
PI(3,5)P
2
PI(4,5)P2
PI(3,4)P2
PI(3,4,5)P3
PI
PI3P
PI4P

brane. To exclude this possibility, we performed an
in vitro binding assay using purified Noxo1b and
Noxo1c. As shown in Fig. 7A, Noxo1c-DC and
Noxo1b-DC bound to the C-terminus of p22
phox
to the
same extent; the binding was completely abolished by
the P156Q substitution in p22
phox
, a mutation leading
to defective interaction with the SH3 domains of
Noxo1 [15]. In addition, Noxo1c as well as Noxo1b
interacted with p22
phox
in a similar manner in the yeast
two-hybrid system (Fig. 7B). Thus the insertion in the
PX domain does not seem to affect the SH3-mediated
interaction with p22
phox
. To confirm this, we investi-
gated the dependence of Noxo1c-supported Nox
activation on p22
phox
. It is known that superoxide pro-
duction by Nox1 in CHO cells expressing Noxo1b is
largely but not completely dependent on the cotrans-
fection with the p22
phox
cDNA [15], whereas Nox3
activity requires p22

abrogates the phosphoinositide-binding activity [19].
As shown in Fig. 9A, a mutant Noxo1b carrying the
R40Q substitution failed to support the superoxide
production by Nox1. Thus the PX-mediated lipid
binding is required for Nox1 activation. The R40Q
substitution in Noxo1c also abolished superoxide pro-
duction by Nox1 (Fig. 9A), supporting the conclusion
that Noxo1c retains considerable lipid-binding activity
(Fig. 5B). On the other hand, in Nox3 activation, the
mutant Noxo1 proteins were threefold less active than
the wild-type one (Fig. 9B), suggesting that PX-medi-
ated binding to phosphoinositides is involved in, but
not absolutely required for, Nox3 activity. This is in
contrast with the observation that the PX domain by
itself is essential for Nox3 activation (Fig. 8C). The
partial dependence on the lipid-binding activity may
explain why Noxo1c with a weak but significant
lipid-binding activity (Fig. 5) is equivalent to Noxo1b
in Nox3 activation (Fig. 6). The idea may be suppor-
ted by the observation that a part of Noxo1c as well
as Noxo1b was localized to ruffling membranes in the
Nox3-transfected CHO cells (data not shown).
A
DC
B
Nox3 + Noxa1
ecnecsenimulimehc
0
1
x

6
)mpc
5
3
0
1
4
2
Noxo1
β
β
Noxo1
γ
γ
Nox3
1.2
ecnecsenimulimehc
01x(
7
)mpc
0
0.4
0.8
PMA(–)
PMA(+)
Noxo1
β
β
Noxo1
γ

, pEF-BOS–myc-Noxa1, and simultaneously with pEF-BOS–
HA-Noxo1b or pEF-BOS–HA-Noxo1c in (B); pcDNA3.0–Nox3 and
pEF-BOS–p22
phox
, and simultaneously with pEF-BOS–HA-Noxo1b
or pEF-BOS–HA-Noxo1c in (C); pcDNA3.0–Nox3 and pEF-BOS–
p22
phox
, pEF-BOS–myc-Noxa1, and simultaneously with pEF-BOS–
HA-Noxo1b or pEF-BOS–HA-Noxo1c in (D). Superoxide production
was assayed by chemiluminescence using Diogenes in the
presence or absence of PMA (200 ngÆmL
)1
). Each graph represents
the mean ± SD of the peak chemiluminescence values obtained
from three independent transfections. Protein levels of Noxo1b and
Noxo1c in the transfected cells were estimated by immunoblot
analysis with the monoclonal antibody to HA (lower panels).
R. Takeya et al. Expression and function of Noxo1c
FEBS Journal 273 (2006) 3663–3677 ª 2006 The Authors Journal compilation ª 2006 FEBS 3671
Concluding remarks
In this study, we show that Noxo1c, a novel alternat-
ive splicing form of human Noxo1 containing an addi-
tional five amino acids in the PX domain, is expressed
in the testis and fetal brain (Fig. 2). During the
revision of this manuscript, Cheng & Lambeth [28]
reported the expression and function of the four splice
forms of human Noxo1. The Noxo1c mRNA is also
expressed in several Nox1-expressing and Noxa1-
expressing human cancer cell lines [the androgen-

2
0
3
1
ec
n
ecsenimu
l
imehc
01x(
5
)mpc
Nox3 + Noxo1
γ
γ
e
cnecsenimulimehc
01x(
6
)mpc
2.0
1.2
0
0.4
1.6
0.8
Nox1 + Noxa1 + Noxo1
γ
γ
– p22

-
Δ
C
His
(+) (–)
Noxo1
β
-
Δ
C
p22
phox
-C
(WT)
p22
phox
-C
(P156Q)
His
(+) (–)
1oxoN–TSG
β
-
Δ
C
1oxoN–TSG
γ
-
Δ
C

in (C); pcDNA3.0–Nox3, pEF-BOS–
myc-Noxa1, pEF-BOS–HA-Noxo1c, and with or without pEF-BOS–p22
phox
in (D). Superoxide production was assayed by chemiluminescence
using Diogenes in the presence of PMA (200 ngÆmL
)1
). Each graph represents the mean ± SD of the peak chemiluminescence values
obtained from three independent transfections.
Expression and function of Noxo1c R. Takeya et al.
3672 FEBS Journal 273 (2006) 3663–3677 ª 2006 The Authors Journal compilation ª 2006 FEBS
GGCAGGCCCCCGATACCCAG-3¢ and 5¢-CGTCTCGA
G
GAGGCGGCCCGCAGCGCGAGA-3¢; sequences from
the mRNA are underlined. With the two primers, PCR was
performed using Human Multiple Tissue cDNA (MTC
TM
)
panels (Clontech, Mountain View, CA, USA) as a template,
and the PCR products were subcloned into pBluescript.
Sequencing analysis of the PCR products corroborated
four previously reported variants: Noxo1b (AB097667,
AF532984, and AF539796), Noxo1c (AF532985), Noxo1a
(AY255768 and AF532983), and Noxo1d (AY191359). On
the basis of the sequence of splice variants, we synthesized
the full-length Noxo1b, Noxo1c, Noxo1a, and Noxo1d by
PCR-mediated site-directed mutagenesis, and the DNA
fragments were cloned into vectors. All the constructs were
sequenced to confirm their identities.
A
gp91

4.0
0
3.0
2.0
1.0
ecn
ecse
ni
m
uli
me
hc
01x
(
6
)mpc
3.0
0
2.0
1.0
Noxo1
β Δ
PX vector
Noxo1
β Δ
PX vector
Fig. 8. Role of the Noxo1 PX domain in activation of Nox enzymes.
CHO cells were cotransfected with the following combination
of plasmids: pcDNA3.0–Nox1, pEF-BOS–p22
phox

γ
(R40Q)
Noxo1
γ
Noxo1
β
A
Nox1 + Noxa1
ec
necsenimulimeh
c
01x(
5
)
m
pc
8
4
0
6
2
Noxo1
β
(R40Q)
Noxo1
γ
(R40Q)
Noxo1
γ
Noxo1

human tissues
The expression patterns of the Noxo1b and Noxo1c mes-
sengers were determined by PCR using Human Multiple
Tissue cDNA panels and Human Fetal Neural Tissue
cDNA panels (Biochain Institute, Hayward, CA, USA),
according to the manufacturer’s protocol. Expression of
Noxo1c was determined by RT-PCR using total RNA as a
template, which was extracted by TRIzol reagent (Invitro-
gen, Carlsbad, CA, USA) from the following human cell
lines; androgen-independent prostate cancer LNCaP cells,
androgen-independent prostate cancer PC3 and DU145
cells, and testicular germ cell tumor NEC8 cells [38,39].
Splicing-specific PCR was performed using the following
primers: ‘a’, 5¢-TCTCCCAAAGCTTCTCGATGC-3¢ (for-
ward primer specific for the Noxo1 cDNA); ‘b’,
5¢-CCCAAAGCTTCTCGGTCAGGC-3¢ (forward primer
specific for the Noxo1c cDNA); ‘c’, 5¢-TCTGGGGTGGG
CAGGATCACC-3¢ (reverse primer for both the Noxo1b
and Noxo1c cDNA). To amplify both the Noxo1b and
Noxo1c cDNAs, the following primer, ‘d’, 5¢-CCGCGT
TCTCCCAAAGCT-3¢ and primer ‘c’ were used. 5¢-GA
AATCCCATCACCATCTTCCA-3¢ (forward primer) and
5¢-CCTTCTCCATGGTGGTGAAGAC-3¢ (reverse primer)
were used for the glyceraldehyde-3-phosphate dehydroge-
nase cDNA. PCR analyses were performed using ABI
PRISMÒ 9700 (Applied Biosystems, Foster City, CA, USA)
according to the manufacturer’s instructions. The reaction
mixture (10 lL) contained KOD-plus DNA polymerase
(Toyobo, Osaka, Japan), 0.3 lm each primer, and 2 lLof
the first-strand cDNA from different human tissues

phox
as a protein without a tag. Transfection
of the CHO cells with the cDNAs was performed using
FuGENE6 Transfection Reagent (Roche Diagnostics,
Mannheim, Germany). After culture for 30 h, adherent
cells were harvested by incubating with trypsin ⁄ EDTA
for 1 min at 37 °C, and washed with Hepes-buffered sal-
ine (120 mm NaCl, 5 mm KCl, 5 mm glucose, 1 mm
MgCl
2
, 0.5 mm CaCl
2
and 17 mm Hepes, pH 7.4). Super-
oxide production by the transfected cells was determined
by superoxide dismutase-inhibitable chemiluminescence
with an enhancer-containing luminol-based detection system
(Diogenes; National Diagnostics, Atlanta, GA, USA), as pre-
viously described [15,23,40,41]. After the addition of the
enhanced luminol-based substrate, the cells were stimulated
with 200 ngÆmL
)1
PMA. The chemiluminescence was
assayed using a luminometer (Auto Lumat LB953; Berthold
Technologies, Bad Wildbad, Germany).
Measurement of superoxide production using
adherent cells undetached from culture dishes
CHO cells were plated on six-well plates (1 · 10
5
cells ⁄ well)
18 h before the transfection. Cells were transfected with

2
PO
4
), and blocked with
NaCl ⁄ P
i
containing 3% BSA for 60 min. The sample was
subsequently incubated with the monoclonal antibody to
HA and probed with Alexa Fluor 488
TM
-labeled goat
anti-mouse IgG (Invitrogen, Carlsbad, CA, USA) as sec-
ondary antibodies. For detection of p22
phox
, the sample
was incubated with polyclonal antibodies to p22
phox
,
which were raised against the C-terminal 20 amino acids
of human p22
phox
[25] and probed with Alexa Fluor
Expression and function of Noxo1c R. Takeya et al.
3674 FEBS Journal 273 (2006) 3663–3677 ª 2006 The Authors Journal compilation ª 2006 FEBS
594
TM
-labeled goat anti-rabbit IgG (Molecular Probes) as
secondary antibodies. Images were visualized with a con-
focal laser-scanning microscope LSM5 PASCAL (Carl
Zeiss, Oberkochen, Germany).

brane fraction. Proteins were analyzed by western blot with
the monoclonal antibody to HA and developed by using
ECL-plus.
Lipid-binding assay using recombinant
GST–fusion proteins
The PX domain of Noxo1b (amino acids 1–153) and its
corresponding region of Noxo1c (amino acids 1–158) were
expressed as proteins fused to GST in Escherichia coli strain
BL21, and purified by glutathione–Sepharose 4B (Amer-
sham Bioscience), as previously described [14,21].
An overlay assay was carried out using the PIP array
TM
(Echelon Biosciences, Salt Lake City, UT, USA) following
the manufacturer’s protocol. Membranes were first incuba-
ted with 4% nonfat dry milk in Tris-buffered saline ⁄ Tween
(20 mm Tris ⁄ HCl, pH 7.5, 136 mm NaCl, 0.1% Tween-20)
at room temperature for 1 h and then overnight at 4 °C
with 500 ngÆmL
)1
GST fusion protein. After being washed
three times with Tris-buffered saline ⁄ Tween, the membranes
were incubated with 1 : 1000 goat polyclonal antibodies to
GST (Amersham Bioscience). Membranes were further incu-
bated with 1 : 2500 donkey anti-goat IgG conjugated to
horseradish peroxidase (Santa Cruz Biotechnology, Santa
Cruz, CA, USA). The antibodies were detected by chemilu-
minescence using ECL-plus as previously described [15].
In vitro liposome-binding assay was carried out as
previously described [20,23] with minor modifications.
Briefly, liposomes were prepared by mixing phosphatidyl-

Various combinations between pGBT9 (Clontech) and
pGADGH (Clontech) plasmids, each encoding an oxidase
protein, were cotransformed into competent yeast HF7c
cells containing a HIS3 reporter gene, as previously des-
cribed [15]. After the selection for Trp
+
and Leu
+
pheno-
type, the transformants were tested for their ability to grow
on plates lacking histidine, according to the manufacturer’s
recommendation (Clontech).
Acknowledgements
We are grateful to Yohko Kage (Kyushu University
and JST), Miki Matsuo (Kyushu University), Natsuko
Yoshiura (Kyushu University), and Namiko Kubo
(Kyushu University and JST) for technical assistance,
and to Minako Nishino (Kyushu University and JST)
for secretarial assistance. This work was supported in
part by Grants-in-Aid for Scientific Research and
National Project on Protein Structural and Functional
Analyses from the Ministry of Education, Culture,
Sports, Science and Technology of Japan, and CREST
and BIRD projects of JST (Japan Science and
Technology Agency).
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