Enzymatic actions of Pasteurella multocida toxin detected
by monoclonal antibodies recognizing the deamidated
a subunit of the heterotrimeric GTPase G
q
Shigeki Kamitani
1
, Shinpei Ao
2
, Hirono Toshima
1
, Taro Tachibana
2
, Makiko Hashimoto
1
,
Kengo Kitadokoro
3
, Aya Fukui-Miyazaki
1
, Hiroyuki Abe
1
and Yasuhiko Horiguchi
1
1 Department of Molecular Bacteriology, Research Institute for Microbial Diseases, Osaka University, Japan
2 Department of Bioengineering, Graduate School of Engineering, Osaka City University, Japan
3 Department of Biomolecular Engineering, Graduate School of Science and Technology, Kyoto Institute of Technology, Japan
Introduction
Pasteurella multocida toxin (PMT) is a highly potent
mitogen acting on various types of cultured cells,
including fibroblasts and osteoblastic cells [1–3].
Because of this, it is referred to as cyclomodulin, which

Bacteriology, Research Institute for
Microbial Diseases, Osaka University 3-1
Yamada-oka, Suita-shi, Osaka 565-0871,
Japan
Fax: +81 6 6879 8283
Tel: +81 6 6879 8285
E-mail:
(Received 12 October 2010, revised 9
May 2011, accepted 25 May 2011)
doi:10.1111/j.1742-4658.2011.08197.x
Pasteurella multocida toxin (PMT) is a virulence factor responsible for the
pathogenesis of some Pasteurellosis. PMT exerts its toxic effects through
the activation of heterotrimeric GTPase (G
q
,G
12 ⁄ 13
and G
i
)-dependent
pathways, by deamidating a glutamine residue in the a subunit of these
GTPases. However, the enzymatic characteristics of PMT are yet to be
analyzed in detail because the deamidation has only been observed in cell-
based assays. In the present study, we developed rat monoclonal antibod-
ies, specifically recognizing the deamidated Ga
q
, to detect the actions of
PMT by immunological techniques such as western blotting. Using the
monoclonal antibodies, we found that the toxin deamidated Ga
q
only

q
was
also considered to be deamidated by the toxin. The
deamidated GTPases were found to lose their GTPase
activity and, as a result, stimulate downstream signal-
ing pathways. Taken together, all these findings sug-
gest that the catalytic triad in the C3 domain conducts
the deamidation reaction. However, the enzymatic
characteristics of PMT have not been analyzed as a
result of the lack of an easily-administered assay to
detect activity of the toxin.
In the present study, we developed rat monoclonal
antibodies that specifically recognize the deamidated
a subunit of G
q
(anti-Ga
q
Q209E) and obtained results
providing new insights into the enzymatic actions of
PMT. In addition, the monoclonal antibodies enabled
us to detect PMT-induced deamidation in situ, indicat-
ing them to be powerful probes for characterizing the
actions of the toxin.
Results
Analysis of enzymatic actions of PMT with Ga
q
Q209E-specific monoclonal antibodies
According to a previous study [16], the deamidation of
Ga by PMT results in the conversion of a Gln residue
in the switch 2 region to Glu. To raise antibodies to

FSFKDLNFRMFDVGGQRSERKKWIHCFEG
G
α
t2
FSVKDLNFRMFDVGGQRSERKKWIHCFEG
G
α
i1
FTFKDLHFKMFDVGGQRSERKKWIHCFEG
G
α
i3
FTFKELYFKMFDVGGQRSERKKWIHCFEG
G
α
i2
FTFKDLHFKMFDVGGQRSERKKWIHCFEG
G
α
o1
FTFKNLHFRLFDVGGQRSERKKWIHCFED
G
α
o2
FTFKNLHFRLFDVGGQRSERKKWIHCFED
G
α
z
FTFKELTFKMVDVGGQRSERKKWIHCFEG
G

FVIKKIPFKMVDVGGQRSQRQKWFQCFDG
G
α
13
FEIKNVPFKMVDVGGQRSERKRWFECFDS
* :::.******.:*::*:.**:.
WT Gq peptide IFRMVDVGGQRSERRKWIHC
MUT Gq peptide IFRMVDVGGERSERRKWIHC
A
194 200 210 220
BC
+ mGαq WT
None
+ mGα
q
Q209E
Anti-Gαq
Anti-Gα11
+ mGαq/11 105–113
+ mG
α
11
Anti-β-actin
3G3
3F6
Anti-Gα
q Q209E
MEF
(–)
complemented by

WB:
Fig. 1. Isolation of Ga
q
Q209E-specific antibodies. (A) Alignment of amino acid sequences of the switch 2 region in the a subunits of mouse
heterotrimeric GTPases by
CLUSTALW. Sequences of synthetic oligopeptides for the generation of antibodies are shown at the bottom of the
panel. The sequences corresponding to the oligopeptides are highlighted. The nucleotide sequences are obtained from NCBI; Ga
t1
(accession
number NP_032166), Ga
t2
(NP_032167), Ga
i1
(NP_034435), Ga
i2
(AAH65159), Ga
i3
(NP_034436), Ga
o1
(P18872), Ga
o2
(P18873), Ga
z
(NP_034441), Ga
s
(P63094), Ga
solf1
(NP_034437), Ga
solf2
(NP_796111), Ga

, mutant Ga
q
Q209E, Ga
11
or Ga
q ⁄ 11 105–113
was subjected to 15% SDS ⁄ PAGE and western blotting with monoclonal rat anti-Ga
q
Q209E (3F6 or 3G3), polyclonal rabbit anti-Ga
q
, polyclonal
rabbit anti-Ga
q11
or polyclonal rabbit anti-b-actin. (C) The substrate specificity of rat anti-G a
q
Q209E monoclonal for the key members of Ga su-
bunits. The deamidated forms of each mutant Ga subunits were detected by anti-Ga
q
Q209E (3G3). 293T cells were transfected pEF6-based
plasmids expressing the indicated Ga subunits. After 24 h of incubation, the cells were lysed and subjected to 15% SDS ⁄ PAGE followed by
western blotting with monoclonal rat anti-Ga
q
Q209E (3G3), polyclonal rabbit anti-Ga
s
, polyclonal rabbit anti-Ga
i-2
, polyclonal rabbit anti-Ga
13
,
serum polyclonal rabbit anti-Ga

Q209E-specific anti-
bodies, we attempted to detect the deamidation of the
recombinant Ga
q
caused by in vitro treatment with
PMT or PMT variants under various conditions. In
these experiments, Ga
i ⁄ q
was used in place of Ga
q
because the former chimera was more stable and solu-
ble and more readily prepared than the latter wild-type
[26]. The deamidation of Ga
i ⁄ q
was detected by the
antibody when Ga
i ⁄ q
b
1
c
s
was treated with wild-type
C-PMT and the full-length PMT (Fig. 2A). C-PMT,
which consists of only the intracellularly active domains
[23], appeared to deamidate Ga
i ⁄ q
b
1
c
s

in vitro,
whereas GST-C3 C1165S and GST alone showed no
deamidation activity (Fig. 2D). GST-C3 WT was
approximately 100-fold less efficient than C-PMT.
C-PMT deamidated Ga
i ⁄ q
in both the monomeric
and heterotrimeric state in vitro, although the mono-
meric Ga
i ⁄ q
was approximately 100-fold less sensitive
than the heterotrimeric form (Figs 3A and S1A). Ga
i ⁄ q
was also deamidated when the concentration of mono-
meric Ga
i ⁄ q
increased (Fig. 3B).
Ga
11
as another target for PMT
The sequence of the WT G
q
-peptide is completely con-
sistent with the corresponding region of Ga
11
(Fig. 1A).
Indeed, the deamidated form of Ga
11
(Ga
11

, was insensitive to PMT. We
constructed Ga
q ⁄ 11
-deficient MEF cells expressing
either Ga
11
or the chimeric Ga
q ⁄ 11 105–113
(Fig. 1B) and
examined their sensitivity to PMT. As shown in
Fig. 4A, both Ga
11
and Ga
q ⁄ 11 105–113
were deamidated
by PMT. Furthermore, we examined whether each of
the cells responds to the PMT treatment by determining
intracellular PLC activity (Fig. 4B). In addition to Ga
q
,
Ga
11
and the chimeric Ga
q ⁄ 11 105–113
conferred sensitivity
to PMT on Ga
q ⁄ 11
-deficient MEF cells, although the
magnitude of the response to PMT was small in the cells
expressing Ga

q
, in native gel electro-
phoresis, as detected by Ga
q ⁄ 11
-specific immunoblot
analysis, and the Ga
q
Q209E-specific antibody only
recognized the migration-increased Ga
11
(Fig. S2A).
Furthermore, using immunoprecipitation of Ga
11
and
western blotting, we confirmed that PMT deamidated
Ga
11
expressed in MEF Ga
q ⁄ 11
-deficient cells (Fig. S2B).
Application of Ga
q
Q209E-specific monoclonal
antibodies to detect PMT activity
The Ga
q
Q209E antibodies also detected the deamida-
tion of the endogenous Ga caused by PMT in
Swiss3T3 cells (Fig. 4C), although the subtype of Ga
could not be identified. On the basis of the immuno-

by PMT was identified by MS
[16], that of Ga
i1
and Ga
q
was only supported by indi-
rect evidence, such as the alteration of isoelectric
points demonstrated by 2D or native gel electrophore-
sis [16].
In the present study, we aimed to analyze the enzy-
matic characteristics of PMT by using monoclonal rat
antibodies that specifically recognize the deamidated
Ga
q
. Previously, we succeeded in detecting the small
GTPase Rho deamidated by dermonecrotic toxin from
Bordetella bronchiseptica by using rabbit antibodies
specifically recognizing the deamidated residues [27].
The deamidation catalyzed by PMT and by dermone-
crotic toxin occurs on a Gln residue that is conserved
among GTPases and essential for GTPase activity. We
therefore expected a similar strategy for detecting
PMT-catalyzed deamidation to be successful. Indeed,
we could detect PMT activity both in vitro and in situ
by using the monoclonal antibodies.
75
37
50
MW (kDa)
Gαq Q209E

C-PMT WT
Mock
0.01 0.01
GST-C3
C1165S
GST-C3
WT
0 0.1 1 0.1 10.01 0.01 10.1
50
37
75
25
Protein (μM)
Gα
q Q209E
MW (kDa)
MW (kDa)
C-PMT
WT
Mock
C-PMT
C1165S
C-PMT
C1159S
–+ –+ –+–+
PMT: Gαi/qβ1γs
= 100 nM : 1 μM
Gαi/qβ1γs = 1 μM
Gαi/qβ1γs = 1 μM
A

WB:
CBB:
50
Gαq
Gαq
50
WB:
CBB:
50
WB:
CBB:
50
Gαq
Gαq
Fig. 2. In vitro deamidation of Ga
i ⁄ q
b
1
c
s
by
PMT. Ga
i ⁄ q
b
1
c
s
and PMT or PMT variants
were incubated at 37 °C overnight under
various conditions and subjected to 15%

q ⁄ 11
-
deficient cells expressing Ga
q
Q209E was
used as the positive control. (B) In vitro
deamidation of Ga
i ⁄ q
by PMT under reduc-
ing conditions. Ga
i ⁄ q
b
1
c
s
at 1 lM was incu-
bated with C-PMT at 10 n
M (upper panel) or
100 n
M (lower panel) in the presence or
absence of 5 m
M dithiotreitol. (C) C-PMT
DC1(4H) deamidates Ga
i ⁄ q
in vitro. C-PMT,
C-PMT C1165S, C-PMT C1159S or C-PMT
DC1(4H) at the indicated concentrations and
1 l
M Ga
i ⁄ q

enzymatic actions are known to undergo intracellular
cleavage after binding to specific receptors on target
cells. Similarly, intramolecular cleavage may occur on
PMT and C-PMT encompassing the catalytic domain
may be liberated into the cytoplasm, where the sub-
strates, Ga proteins, reside. Thus, the N-terminal
region of PMT may hamper the action of the cata-
lytic C-PMT. (b) The C3 domain of C-PMT alone
showed the deamidation activity (Fig. 2D). It was pre-
viously reported [24] that C-PMT is the minimum
unit required for intracellular toxicity after transloca-
tion into the cytoplasm. Indeed, when expressed in
cells, C-PMT lacking the C1 domain, which functions
as the membrane-targeting domain [24], no longer
affected the cells. These results indicate that the C1,
C2 and C3 domains must coordinate in the cytoplasm
for the cytotoxicity to occur, although the enzymatic
action is attributable to the C3 domain per se. (c)
C-PMT deamidated the Ga proteins only under
reducing conditions, whereas C-PMT C1159S did so
under both reducing and nonreducing conditions
(Fig. 2B). These results confirm that cleavage of the
disulfide bond between Cys
1159
and Cys
1165
in the C3
domain is essential for formation of the catalytic triad
comprising Cys
1165

was also deamidated by PMT. Moreover, MEF
())
cells
complemented with Ga
q
or Ga
q ⁄ 11 105–113
responded
to PMT with an increase in intracellular inositol phos-
phates, indicating the activation of PLCb downstream
of Ga
q
or Ga
11
. Furthermore, PMT increased the
migration of Ga
11
protein in native gel electrophoresis,
probably as a result of PMT-catalyzed deamidation.
The combination of immunoprecipitation and western
blotting by using the Ga
q
Q209E-specific antibody also
revealed that Ga
11
expressed in MEF
())
cells was
deamidated by PMT (Fig. S2). These results were
inconsistent with the previous observation that Ga

q
and Ga
11
(Fig. 3A).
These cells did not show an increase in inositol phos-
phate levels in response to the toxin and, thus, the
additional substrate could not be upstream of PLCb.
A
Gαq Q209E
10 0.01 0.1 10.01 0.10
75
37
50
MW (kDa)
Gαi/qβ1γs
Gαi/q
C-PMT (μM)
B
75
37
50
MW (kDa)
Gαq Q209E
515 1 5511G protein (μM)
C-PMT
C-PMT
Buffer
Buffer
Gαi/qβ1γs
Gαi/q

i ⁄ q
b
1
c
s
,orGa
i ⁄ q
at 1 and 5 lM was incubated with
10 n
M C-PMT. Recombinant Ga
i ⁄ q
proteins after incubation with
C-PMT were respectively applied at 1.1 or 5.5 lg per each lane,
and recombinant Ga
i ⁄ q
b
1
c
s
were at 4.5 and 22.5 lg per each lane.
In all experiments, the reaction mixture after incubation at 37 °C
overnight was subjected to 15% SDS ⁄ PAGE and western blotting
with rat anti-Ga
q
Q209E (3F6) (upper panel). The loaded recombi-
nant Ga
i ⁄ q
was visualized by Coomasie Brilliant Blue staining (lower
panel).
Enzymatic actions of P. multocida toxin S. Kamitani et al.

-peptide is com-
pletely consistent with or highly homologous to the
corresponding region of Ga
14
or Ga
15
, and the anti-
bodies recognized the Ga
14
Q205E mutant protein in
the lysate of cells expressing mouse Ga
14
Q205E on
A
C
WB:
+PMT
No stimulation
Deamidated Gα
q
Nucleus
None
+ mG
α
q
WT
PMT
– + –+ –+–+ – +–+
+ mGα
11

+ mGα
11 WT
+ mG
α
q/11 105–113
+ mGαq Q209E
Swiss3T3
MEF
(–)
n.s.
n.s.
P = 0.0040
P = 0.0056
P = 0.0174
P = 0.0019
Anti-Gαq Q209E
Anti-Gα
q
10000101001
WB:
100010 1001
MEF
(–)
+ mGαq Q209E
WT C1165S
PMT
(ng/ml)
Anti-β-actin
Anti-β-actin
Anti-Gα

PMT, and intra-
cellular [
3
H]inositol phosphates, which are products of the enzymatic action of PLC, were measured as described in the Experimental
procedures. Each bar represents the mean of triplicate measurements, with the error bar indicating the SD. Representative results from
three independent experiments are shown. The statistical significance of differences between PMT-treated and untreated cells was evalu-
ated by a paired t-test. P < 0.05 was considered statistically significant. (C) PMT deamidated Ga
q
in Swiss3T3 cells. Swiss3T3 cells were
treated with PMT WT or PMT C1165S at the indicated concentrations. After 4 h of treatment, cells were lysed and subjected to 15%
SDS ⁄ PAGE followed by western blotting with monoclonal rat anti-Ga
q
Q209E (3F6) as described in the Experimental procedures. The lysate
of MEF Ga
q ⁄ 11
-deficient cells expressing Ga
q
Q209E was used as the positive control. (D) Immunofluorescent microscopy of Swiss3T3 cells
treated with 100 ngÆmL
)1
PMT for 4 h. After fixing and permeabilization of the cells, the deamidated Ga and the nucleus were visualized
with anti-Ga
q
Q209E 3G3 (green) and 4¢,6-diamidino-2-phenyl-indole (blue), respectively. Images are presented at the same magnification.
S. Kamitani et al. Enzymatic actions of P. multocida toxin
FEBS Journal 278 (2011) 2702–2712 ª 2011 The Authors Journal compilation ª 2011 FEBS 2707
western blotting (Fig. S3B). Taken together, the anti-
bodies could be useful for detecting the PMT-catalyzed
deamidation of Ga proteins. It is noteworthy that they
detected localization of the tissues or cells influenced

11
[29], were used as
the template DNA. Consequently, each amplified DNA
fragment was once cloned into pEF6-V5 ⁄ His-TOPO TA
(Invitrogen, Carlsbad, CA, USA), and then sequenced.
Plasmid clones containing the correct sequence of Ga
q
or
of Ga
11
were respectively designated pEF6-mGa
q
and
pEF6-mGa
11
. pEF6-mGa
q
or pEF6-mGa
11
was excised
with BamHI and NotI and cloned into the BamHI-NotI
site of pCXbsr [30]. The resultant plasmids were desig-
nated pCXbsr-mGa
q
and pCXbsr-mGa
11
. pCXbsr-Ga
q
Q209E and pCXbsr-G a
q ⁄ 11 105–113

and pCMV5-hGa
13
[31] for pEF6-V5 ⁄ His-
hGa
13
were used as the template DNA. Consequently, each
amplified DNA fragment was cloned into pEF6-V5 ⁄ His-
TOPO TA (Invitrogen) and then sequenced. Plasmid clones
containing the correct sequence of each Ga subunit were
designated pEF6-V5 ⁄ His-Ga
s
, pEF6-V5 ⁄ His-Ga
i-2
and
pEF6-V5 ⁄ His-Ga
13
, respectively. All deamidated mutant
plasmids of pEF6-V5 ⁄ His-Ga plasmids were constructed by
a QuikChange II site-directed mutagenesis kit (Stratagene)
with the mutagenized primers listed in Table S1 and pEF6-
V5 ⁄ His-Ga
s
, pEF6-V5 ⁄ His-Ga
i-2
and pEF6-V5 ⁄ His-Gas
13
as the template DNA in accordance with the manufac-
turer’s instructions.
Plasmids for expression in E. coli
pPROEX-1-C-PMT [32], pPROEX-1-C-PMT C1165S [23],

⁄ Ga
11
or Ga
12
⁄ Ga
13
gene-defi-
cient or wild-type mice were cultured as described previ-
ously [10,33,34]. For production of the retroviral vector,
Plat-E cells were transfected with the retroviral transfer vec-
tor used in the plasmid construction by Lipofectamin 2000
(Invitrogen) in accordance with the manufacturer’s instruc-
tions. In brief, 2 · 10
5
cells were seeded in each well of a
six-well plate. The next day, 1.0 lg of the retroviral transfer
vector was transfected. The supernatant was collected after
2 days and centrifuged to spin down cellular debris.
Ga
q
⁄ Ga
11
gene-deficient MEF cells (2 · 10
5
cells) were
infected in the presence of 5 lgÆmL
)1
polybrene (Nacalai
tesq, Kyoto, Japan) after filtration of the virus-containing
medium with a 0.22 lm membrane (Millipore, Billerica,

Fluorescence microscopy
Swiss3T3 cells were seeded into 24-well plates containing
glass coverslips (Matsunami, Osaka, Japan). After incuba-
tion overnight, the cells were treated with 100 nm PMT for
4 h, and fixed with 3.7% formaldehyde in NaCl ⁄ Pi for
15 min. After treatment with 0.1% Triton X-100 in
NaCl ⁄ Pi for 5 min at room temperature and subsequently
with 3% skimmed milk in NaCl ⁄ Pi for 30 min, the cells
were stained with monoclonal rat the Ga
q
Q209E-specific
antibody, 3G3, for 1 h at room temperature. They were
washed with NaCl ⁄ Pi three times, stained with Alexa Flour
488-conjugated anti-rat IgG serum (Invitrogen) for 30 min
at room temperature, washed again with NaCl ⁄ Pi, and
treated with Slow Fade GOLD antifade reagent with
4¢,6-diamidino-2-phenyl-indole (Invitrogen). The cells were
examined under microscopy with an epifluorescence micro-
scope (BX50; Olympus, Tokyo, Japan). Images were cap-
tured and analyzed by SlideBook 4.0 (Roper Industries,
Inc., Sarasota, FL, USA) to control the fluorescent decon-
volution microscopy.
Purification of heterotrimeric Ga
i ⁄ q
b
1
c
s
and
monomeric Ga

has an N-terminal His
6
tag,
followed by a TEV cleavage site, amino acids 1–28 of rat
Ga
i1
, a linker of Arg and Ser, and the 37–359 amino acid
region of mouse Ga
q
. The bovine Gb
1
gene and bovine
His
6
-tagged soluble Gc
2
gene (C68S mutant, henceforth
referred to as Gc
s
) were used for the expression of Gb
1
and
Gc
s
. Baculoviruses were amplified by infection of Sf9 insect
cells [36] in Sf9-SFM select medium in accordance with the
manufacturer’s instructions.
Expression and purification of Ga
i ⁄ q
b

ce homogenizer followed by sonication. The sample was
centrifuged for 40 min at 186 000 g, and the supernatant
was filtered and diluted to a final protein concentration of
5mgÆmL
)1
with buffer A (20 mm Hepes, pH 8.0, 100 mm
NaCl, 1 mm MgCl
2
,50lm GDP and 10 mm b -mercapto-
ethanol) and loaded onto a 10 mL Nickel-NTA column
(Sigma, St Louis, MO, USA) pre-equilibrated with the same
buffer. The column was washed with 200 mL of buffer A
followed by 100 mL of buffer B (buffer A with 300 mm
NaCl and 10 mm imidazole, pH 8.0). Ga
i ⁄ q
b
1
c
s
was eluted
with buffer A supplemented with 150 mm imidazole (pH
8.0). The eluate was dialyzed against buffer A in which
2mm dithiotreitol was substituted for 10 mm b-mercapto-
ethanol. The protein was concentrated using a VIVASPIN2
30 (GE Healthcare) to approximately 1.0 mgÆmL
)1
, and
analyzed by 15% SDS ⁄ PAGE, followed by Coomasie bril-
liant blue staining (Fig. S1A). For preparation of the mono-
meric Ga

The recombinant Ga
i ⁄ q
b
1
c
s
or Ga
i ⁄ q
alone was incubated
with purified recombinant PMT and its mutants at a molar
S. Kamitani et al. Enzymatic actions of P. multocida toxin
FEBS Journal 278 (2011) 2702–2712 ª 2011 The Authors Journal compilation ª 2011 FEBS 2709
ratio of 100 : 1, 10 : 1 or 1 : 1 in 20 mm Tris-HCl (pH 7.5),
10 mm MgCl
2
and 1 mm EDTA with or without 5 mm
dithiotreitol at 37 °C overnight. After incubation, the reac-
tion mixture was subjected to 15% SDS ⁄ PAGE, followed
by western blotting. The deamidated Ga
i ⁄ q
was detected by
the monoclonal rat antibody 3F6, isolated as above.
PLC assay
Swiss3T3 cells were seeded at 5 · 10
4
cellsÆwell
)1
into a
24-well plate and incubated at 37 °C for 2 days. The cells
were washed with inositol-free DMEM (IF-DMEM) twice

sheets (Topseal-A, Packard). The contents were mixed by
shaking for 1 h. The beads were allowed to settle for 2 h,
and the radioactivity of each well was determined using a
TopCount microplate scintillation counter (Packard).
Other materials and methods
The protein concentration in each sample was measured by
Protein Assay CBB Solution (Nacalai Tesque, Kyoto,
Japan) and the Micro BCA Protein Assay Kit (Pierce,
Rockfold, IL, USA). SDS ⁄ PAGE was carried out by the
method of Laemmli [38] in a 15% and a 5–20% gradient
polyacrylamide gel. The 5–20% gradient polyacrylamide gel
was obtained from ATTO (Tokyo, Japan). For western
blotting, the samples in the gel after SDS ⁄ PAGE were elec-
trophoretically transferred onto poly(vinylidene difluoride)
membranes (Bio-Rad Laboratories, Hercules, CA, USA).
The membranes were then treated with 5% skim milk and
the transferred proteins were probed with proper antibodies
and visualized on Fuji Medical film (Fujifilm, Minato-ku,
Japan) with an enhanced chemiluminescence system in
accordance with the manufacturer’s instructions (ECL plus;
GE Healthcare). Antibodies for western blotting were
purchased from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA, USA) for anti-Ga
q
(E-17), anti-Ga
q ⁄ 11
(C-19),
anti-Ga
11
(D-17), anti-Ga

i-2
from Dr M. I. Simon (California
Institute of Technology, CA, USA), of Ga
13
from Dr
H. Itoh (Nara Institute of Science and Technology)
and of Ga
q ⁄ 11
-deficient MEF cells from Drs S. Offer-
manns and B. Zimmermann (University of Heidelberg,
Heidelberg, Germany). We would like to thank Ms
Tomoko Suzuki for secretarial assistance. This work
was supported in part by Grants-in-aid for Scientific
Research from the Ministry of Education, Culture, Sci-
ence and Technology of Japan.
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present study.
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Enzymatic actions of P. multocida toxin S. Kamitani et al.
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