Inhibitory effects of nontoxic protein volvatoxin A1
on pore-forming cardiotoxic protein volvatoxin A2
by interaction with amphipathic a-helix
Pei-Tzu Wu
1
, Su-Chang Lin
2
, Chyong-Ing Hsu
1
, Yen-Chywan Liaw
2
and Jung-Yaw Lin
1
1 Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan
2 Institute of Molecular Biology Academia S inica, Taipei, Taiwan
Volvatoxin A (VVA) has been isolated from Volvari-
ella volvacea, and consists of volvatoxin A2 (VVA2)
and volvatoxin A1 (VVA1) [1]. VVA has several biolo-
gical activities, such as: (a) lysis of human red blood
cells; (b) swelling tumor cells and the mitochondria
of liver cells; (c) inhibition of protein biosynthesis;
and (d) causing cardiac arrest via activation of the
Ca
2+
-dependent ATPase enzyme in the ventricular
microsomal fraction [1–3]. The hemolytic activity of
VVA2 is totally inhibited by VVA1 at a molar ratio of
2 [4,5]. Previous studies have shown that VVA2 is a
b-pore-forming toxin, with a heparin-binding site
(HBS) encoded within the C-terminal b-strands (b6, b7
and b8). This HBS structure is indispensable for the

A1 contains 393 amino acid residues that closely resemble a tandem repeat
of volvatoxin A2. Volvatoxin A1 contains two pairs of amphipathic a-heli-
ces but it lacks a heparin-binding site. This suggests that volvatoxin A1
may interact with volvatoxin A2 but not with the cell membrane. By using
confocal microscopy, it was demonstrated that volvatoxin A1 could not
bind to the cell membrane; however, volvatoxin A1 could inhibit binding
of volvatoxin A2 to the cell membrane at a molar ratio of 2. Via peptide
competition assay and in conjunction with pull-down and co-pull-down
experiments, we demonstrated that volvatoxin A1 and volvatoxin A2 may
form a complex. Our results suggest that this occurs via the interaction of
one molecule of volvatoxin A1, which contains two amphipathic a-helices,
with two molecules of volvatoxin A2, each of which contains one amphi-
pathic a-helix. Taken together, the results of this study reveal a novel
mechanism by which volvatoxin A1 regulates the cytotoxicity of volvatoxin
A2 via direct interaction, and potentially provide an exciting new strategy
for chemotherapy.
Abbreviations
FITC, fluorescein isothiocyanate; GSH, glutathione; GSP, gene-specific primer; HBS, heparin-binding site; RBC, red blood cell; VVA,
volvatoxin A; VVA1, volvatoxin A1; VVA2, volvatoxin A2; VVA1-CTD, volvatoxin A1 C-terminal domain (198–391 residues); VVA1-NTD,
volvatoxin A1 N-terminal domain (1–197 residues).
3160 FEBS Journal 273 (2006) 3160–3171 ª 2006 The Authors Journal compilation ª 2006 FEBS
membrane interaction of VVA2 [6]. Furthermore, the
VVA2-binding receptor on the cell membrane has been
shown to be a sulfated glycosaminoglycan, as demon-
strated by affinity column chromatography [6]. Binding
of VVA2 to the cell membrane induced a protein con-
formational change of the VVA2 amphipathic a-helices
to form a prepore complex [7–10]. Therefore, the
amphipathic a-helices play an important role in VVA2
oligomerization and pore formation [6].

that determined previously by protein sequencing (sup-
plementary Fig. S1) [5]. Interestingly, the amino acid
sequence of VVA1 was very similar to that of a tan-
dem repeat of VVA2. The N-terminal half fragment of
VVA1, designated volvatoxin A1 N-terminal domain
(VVA1-NTD) (1–197 residues), had 46.3% similarity
with VVA2 (Fig. 1A), whereas the C-terminal frag-
ment, designated volvatoxin A1 C-terminal domain
(VVA1-CTD) (198–391 residues), displayed 49.2%
similarity to VVA2 (Fig. 1A). The similarity between
VVA1-NTD and VVA1-CTD is 42.6%. The tertiary
structure of VVA2 shows that it has a pair of amphi-
pathic a-helices, denoted a-helix-C and a-helix-D [24],
which are essential for VVA2 dimerization [6]. Interest-
ingly, VVA1 also contains a pair of amphipathic
a-helices similar to VVA2 (Fig. 1A) (supplementary
Fig. S2). It has been shown that the amphiphilicity of
the amphipathic a-helix of VVA2 is indispensable for
protein interaction and oligomerization [6]. Secondary
structure analysis of VVA1 suggests that there might
be two pairs of amphipathic a-helices in both the
N-terminal and C-terminal domains. The hydrophobic
moments of amphipathic a-helix-C and a-helix-D of
VVA1-NTD were calculated to be 0.4 and 0.57,
respectively, while those of amphipathic a-helix-D¢ and
a-helix-E¢ of VVA1-CTD were 0.49 and 0.57 [25].
VVA2 has a basic HBS at its C-terminus that is
located within its b-strand, is indispensable for binding
to cell membranes, and has a pI value of 9.6, similar
to that of the snake venom cardiotoxin [6,20–22]. Nei-

RBCs (Fig. 2A, column 2). Strikingly, VVA1 com-
pletely abolished the hemolytic activity of VVA2 at
P T. Wu et al. VVA1 is a novel toxin regulator of VVA2
FEBS Journal 273 (2006) 3160–3171 ª 2006 The Authors Journal compilation ª 2006 FEBS 3161
A
B
liposomes
VVA2
+
+–
–
–
––
–
–
+
S
12345678
PSP SP
+
++
++
+
VVA1
(MkDa)
(4µg)
(4µg)
200
116
97

of VVA2) and various amounts of VVA1 at
37° C for 24 h. Similar to the results obtained in
hemolytic experiments, VVA1 itself had no cytotoxicity
(Fig. 2B, column 2), but was able to inhibit the cyto-
toxicity of VVA2 completely at a VVA2 ⁄ VVA1 molar
ratios of 2 or lower (Fig. 2B, columns 3–5). Further-
more, the cytotoxicity of VVA2 was reactivated at a
molar ratio of 4 when it was incubated with HeLa cells
(Fig. 2B, column 6).
Additionally, confocal microscopy was employed to
study the inhibitory effects of VVA1 on VVA2. The
results showed that VVA1 by itself was unable to bind
to cell membranes (Fig. 3, panel FITC-VVA1 and pan-
els a–d). Moreover, preincubation of VVA2 and VVA1
(at a molar ratio of 2) inhibited VVA2 binding to the
cell membrane (Fig. 3, panels e–h). These results
strongly suggest that VVA1 inhibits the cytotoxicity of
VVA2 by preventing the binding of VVA2 to the cell
membrane.
Interactions between VVA1 and VVA2
To find whether direct interaction between VVA1 and
VVA2 is required for the inhibitory effects of VVA1 on
the pore-forming activity of VVA2, pull-down experi-
ments were performed. As a preliminary experiment, we
investigated the effects of different buffer constituents
on the oligomerization of VVA2. Only Triton X-100,
and not deoxycholate as had been reported for Bcl-2
family members, was able to induce the oligomerization
of VVA2 (supplementary Fig. S3A) [26]. Interestingly,
not even the harsh, denaturing environment of electro-

Fig. 2. Effects of volvatoxin A1 (VVA1) on the hemolytic and cyto-
toxic activity of volvatoxin A2 (VVA2). (A) The hemolytic activity of
VVA2 regulated by VVA1. VVA2 (45 n
M) and various concentrations
of VVA1 were preincubated as indicated, and the percentage of
hemolysis was calculated as described in Experimental procedures.
Each value represents the mean ± SD of three independent experi-
ments. (B) VVA2 cytotoxicity was affected by VVA1. HeLa cells
were treated with VVA2 (17 n
M) and various amounts of VVA1 at
37 °C for 24 h. Cell death was assayed by using a trypan blue
exclusion assay [41]. Means ± SD are shown for three independent
experiments.
P T. Wu et al. VVA1 is a novel toxin regulator of VVA2
FEBS Journal 273 (2006) 3160–3171 ª 2006 The Authors Journal compilation ª 2006 FEBS 3163
via SDS ⁄ PAGE analysis (Fig. 4A, lane 2). Interest-
ingly, again no oligomers of VVA2 were detected after
incubation of VVA2 with VVA1 at a molar ratio of 2
(Fig. 4A, lane 2).
When VVA1 beads were incubated with VVA2,
VVA2 oligomers were adsorbed and eluted (Fig. 4A,
lane 4). Additionally, when VVA1 beads were incuba-
ted with the mixture of VVA2 and VVA1 (molar ratio
2 : 1), VVA2 and VVA1 were detected, but again, no
VVA2 oligomer was found (Fig. 4A, lane 5). Taken
together, these results strongly support the notion that
there is a direct interaction between VVA1 and VVA2,
and that at a VVA2 ⁄ VVA1 molar ratio of 2, VVA1 is
able to inhibit the oligomerization of VVA2. Extending
these results, we hypothesized that the inhibition of

at 37 °C for 30 min in 0.02% Triton X-100. The beads were washed, and the bound proteins were eluted. The protein eluents were identi-
fied by 10% SDS ⁄ PAGE and visualized by silver staining. (B) Co-pull-down experiments. Linear oligomeric VVA2 (VVA1 beads) was prepared
from VVA1 beads, which were treated with VVA2 in 0.02% Triton X-100 buffer, and VVA1 (VVA2 beads) was prepared from VVA2 beads,
which were treated with VVA1 in the same buffer as described in Experimental procedures. The linear oligomeric VVA2 (VVA1 beads) was
then incubated with various amounts of VVA1, while the VVA1 (VVA2 beads) was incubated with various amounts of VVA2. The reaction
products were eluted with 0.5% SDS loading buffer, and the proteins in the eluents were analyzed by 10% SDS ⁄ PAGE and visualized by
silver staining.
P T. Wu et al. VVA1 is a novel toxin regulator of VVA2
FEBS Journal 273 (2006) 3160–3171 ª 2006 The Authors Journal compilation ª 2006 FEBS 3165
identified as necessary for the oligomerization of
VVA2 [6]. Furthermore, we had determined that
VVA1 encoded two regions that displayed a reason-
ably high degree of conservation to the VVA2 olig-
omerization domain. Thus, we hypothesized that
VVA1 may contain two binding sites for complex
formation with VVA2. To address this issue, co-pull-
down experiments were carried out. First, VVA1-
linked beads (VVA1 beads) were incubated with 45 nm
VVA2 [referred to as linear oligomeric VVA2 (VVA1
beads)]. This mixture was then incubated with various
amounts of VVA1 in the presence of 0.02% Triton
X-100, and eluted with 0.5% SDS buffer (Fig. 4B).
The results demonstrated that no VVA1 could be
bound to a VVA1 bead that was saturated with VVA2
oligomers (Fig. 4B, lanes 1–4), which may imply that
one molecule of VVA2 has one binding site for
interacting with either VVA1 or VVA2. Additional
investigation of the characteristics of binding of VVA2
to VVA1 will be necessary to further understand this
important interaction.

NTD-aH-C-D) at a reVVA1-NTD-aH-C-D ⁄ VVA2
molar ratio of 10 (Fig. 5B, lanes 1–3). Interestingly,
the peptide fragment containing the C-terminal helix
pair (reVVA1-CTD-aH-D¢-E¢) was able to efficiently
compete with binding of VVA2 to the bead-linked
VVA1 at a reVVA1-CTD-aH-D¢-E¢⁄VVA2 molar ratio
of 2.5 (Fig. 5B, lanes 4–6). Furthermore, the reHBSF
peptide fragment could not compete with the interac-
tion of VVA2 with VVA1 beads (Fig. 5B, lanes 7–9).
This was an expected result, as the HBS fragment in
VVA2 was identified as the membrane-binding domain
[6]. These results suggest that the N-terminal and the
C-terminal pair of a-helices of VVA1 can bind to
VVA2 independently of each other and thus enable the
direct binding by one molecule of VVA1 of two mole-
cules of VVA2. This further complements our previous
results suggesting an optimal molar ratio of 2 for bind-
ing of VVA2 to VVA1. The anti-VVA2 IgG used in
this experiment only detects VVA2 and does not cross-
react with VVA1 (supplementary Fig. S4).
In the present study, we have shown that VVA1 com-
pletely inhibits the biological activity of VVA2 in vitro
at VVA2 ⁄ VVA1 molar ratios 2 or lower. This begs the
question of why a mushroom would produce a toxin
and at the same time an antidote. We believe that the
major reason why Volvariella volvacea produces VVA1
is so that it can associate with and, at the right ratio,
enhance the toxicity of VVA2. As shown previously, the
LD
50

main characteristics of the toxin during the transport
process in vivo [32,36]. Therefore, a targeted VVA1–
VVA2 complex may be introduced to the host as a
VVA1 is a novel toxin regulator of VVA2 P T. Wu et al.
3166 FEBS Journal 273 (2006) 3160–3171 ª 2006 The Authors Journal compilation ª 2006 FEBS
protoxin, thus having no toxicity to the animal, but with
the ability to target a tumor. Once at the appropriate
site, the VVA1 molecule could be dissociated, allowing
the VVA2 molecules to oligomerize and reactivate their
cytotoxic pore-forming activity. This has been demon-
strated previously, when a mutated anthrax protoxin
was cleaved by urokinase plasminogen activator and
selectively killed a subset of cancer cells that highly
expressed plasminogen activator [31,37–39]. Thus, this
description of a naturally occurring inhibitor of VVA2
represents a significant discovery, although its import-
ance in a clinical setting remains to be investigated.
Experimental procedures
Materials
Taq DNA polymerase and the pGEM-T vector were
obtained from Promega (Madison, WI). Restriction endo-
nucleases and T4 DNA ligase were from New England
Biolabs Inc. (Beverly, MA). The Marathon cDNA amplifi-
cation kit was from Clontech (Palo Alto, CA). Fluorescent
Alexa-568-labeled goat anti-rabbit and fluorescein isothio-
cyanate (FITC) were purchased from Chemicon Inter-
national (Temecula, CA). CNBr-activated Sepharose 4B,
glutathione (GSH)-agarose-4B column and pGEX-2T vec-
tor were from Amersham Biosciences (Uppsala, Sweden).
All other chemicals were of analytical grade.

)-rich
mRNA was reverse-transcribed with a Marathon cDNA
amplification kit [19,40]. The cDNAs were ligated to
Marathon adaptors for 5¢ and 3¢ rapid amplification of
cDNA ends (RACE), and the products were used as the
template for subsequent PCR. In the first PCR, VVA1
cDNA was amplified with the sense degenerate primer A
and the antisense degenerate primer B, corresponding to
amino acid residues 1–6 and 385–391 of VVA1, respect-
ively. The amplified first PCR products were used as tem-
plate for nested PCR with the sense degenerate primer A
and the antisense degenerate primer C, which corresponds
to amino acid residues 163–168 of VVA1. The products
of this second PCR were sequenced and used to design
the specific antisense primers GSP-1, corresponding to
amino acid residues 40–47 of VVA1, and GSP-3, corres-
ponding to amino acid residues 24–31 of VVA1. In the
third PCR, GSP-1 was used along with the Marathon
primer AP-1, and the products were used as the template
for the fourth PCR, in which GSP-3 was used along with
the sense primer AP-2 to obtain the 5¢-end of the VVA1
cDNA.
The products of the second PCR were used to design
GSP-2 and GSP-4 specific sense primers corresponding to
amino acid residues 128–136 and 155–162 of VVA1,
respectively. The two primers were used along with the
Marathon primers AP-1 and AP-2 to yield the 3¢-end of the
VVA1 cDNA.
The full-length VVA1 cDNA was obtained by amplifying
V. volvacea cDNAs with the sense primer GSP-5, which

50% hemolysis) and various amounts of VVA1 were pre-
mixed in NaCl ⁄ P
i
, and then 0.1 mL of human RBCs
(3 · 10
7
cellsÆmL
)1
) was added. The reaction mixtures were
further incubated at 37 °C for 30 min, and the reaction
was terminated by centrifuging at 13 000 g for 5 min
(KUBOTA RA-155; Kubota, Osaka, Japan). The superna-
tant was measured at 540 nm to determine the degree of
hemolysis. One hundred per cent hemolysis was defined as
the same volume of the human red blood cells in the pres-
ence of 0.1% Triton X-100 [6].
Cytotoxicity assay
HeLa cells were grown in DMEM supplemented with 10%
FBS, 2 mml-glutamine, 100 unitsÆmL
)1
penicillin, and
100 lgÆmL
)1
streptomycin (Life Technologies, Inc.) under
5% CO
2
at 37 °C. HeLa cells (3 · 10
5
) were then treated
with mixtures of VVA2 (17 nm, causing 50% cytotoxicity)

) for 30 min. The cells were then probed with
anti-VVA2 (1 : 1000) at room temperature for 1 h. After
extensive washing in NaCl ⁄ P
i
, the washed cells were stained
with Alexa-568-conjugated goat anti-rabbit IgG (1 : 1000)
for 60 min. During the last 15 min of secondary antibody
staining, Hoechst 33258 (5 lgÆmL
)1
) was applied for observa-
tion of the nucleus. After washing with NaCl ⁄ P
i
, slides were
mounted in mounting solution (80% glycerol in NaCl ⁄ P
i
),
and sealed with nail polish. The cells were subjected to
VVA1 is a novel toxin regulator of VVA2 P T. Wu et al.
3168 FEBS Journal 273 (2006) 3160–3171 ª 2006 The Authors Journal compilation ª 2006 FEBS
immunostaining for observation of the VVA1 and VVA2 as
described previously [41].
Pull-down experiment
VVA2, VVA1 or BSA (8 mg) was coupled to CNBr-Seph-
arose beads (Amersham Pharmacia Biotech) in coupling
buffer (100 mm NaHCO
3
, pH 8.3, and 500 mm NaCl) and
incubated at 4 °C overnight. Residual active groups were
blocked with 1 m ethanolamine, pH 8.0, at room tempera-
ture for 2 h. The beads were then washed four times with

to 10% SDS ⁄ PAGE analysis.
Peptide competition assay
To determine the protein–protein interaction site for VVA1
and VVA2, the amphipathic a-helix regions of VVA1-NTD
(72–109 residues of VVA1) and VVA1-CTD (260–302 resi-
dues of VVA1) were PCR amplified (supplementary Table
S1), and then ligated into the pGEX-2T vector for protein
expression. The HBS fragment of VVA2 (165–199 residues
of VVA2) was constructed as described previously [6]. The
GST fusion proteins were expressed in Escherichia coli and
purified by affinity chromatography on a GSH-agarose-4B
column, and this was followed by thrombin digestion to
obtain pure peptide fragments of VVA1-NTD-aH-C-D,
VVA1-CTD-aH-D¢-E¢ or VVA2 (HBS fragment). For the
competition assay, the various amounts of peptide compet-
itor were added to the mixture of VVA2 and VVA1 (at a
molar ratio of 2), and the mixture was incubated with
VVA1 beads at 37 °C for 30 min, and then washed and
eluted as described above. The eluent was separated by
SDS ⁄ PAGE and transferred to the polyvinylidene difluo-
ride membrane, and western blots were prepared using
anti-VVA2 as a standard protocol [6].
Acknowledgements
We would like to thank Professor Ta-Hsiu Liao for
his valuable suggestions, and Professor Zee-Fen
Chang, Laura Heraty and Dr Brett Hosking for their
critical reading of this manuscript. This work was sup-
ported in part by Grant NSC 89-2320-B-002-238 and
Grant NSC 93-2320-B-002-107 from the National
Science Council, Republic of China.

FEBS Journal 273 (2006) 3160–3171 ª 2006 The Authors Journal compilation ª 2006 FEBS 3169
8 Hotze EM, Heuck AP, Czajkowsky DM, Shao Z,
Johnson AE & Tweten RK (2002) Monomer–monomer
interactions drive the prepore to pore conversion of a
beta-barrel-forming cholesterol-dependent cytolysin.
J Biol Chem 277, 11597–11605.
9 Czajkowsky DM, Hotze EM, Shao Z & Tweten RK
(2004) Vertical collapse of a cytolysin prepore moves its
transmembrane beta-hairpins to the membrane. EMBO
J 23, 3206–3215.
10 Bayley H, Jayasinghe L & Wallace M (2005) Prepore
for a breakthrough. Nat Struct Mol Biol 12, 385–386.
11 Valeva A, Palmer M & Bhakdi S (1997) Staphylococcal
alpha-toxin: formation of the heptameric pore is
partially cooperative and proceeds through multiple
intermediate stages. Biochemistry 36, 13298–13304.
12 Song L, Hobaugh MR, Shustak C, Cheley S, Bayley H
& Gouaux JE (1996) Structure of staphylococcal alpha-
hemolysin, a heptameric transmembrane pore. Science
274, 1859–1866.
13 Choe S, Bennett MJ, Fujii G, Curmi PM, Kantardjieff
KA, Collier RJ & Eisenberg D (1992) The crystal
structure of diphtheria toxin. Nature 357, 216–222.
14 Malovrh P, Viero G, Serra MD, Podlesek Z, Lakey JH,
Macek P, Menestrina G & Anderluh G (2003) A novel
mechanism of pore formation: membrane penetration
by the N-terminal amphipathic region of equinatoxin.
J Biol Chem 278, 22678–22685.
15 Milne JC, Furlong D, Hanna PC, Wall JS & Collier RJ
(1994) Anthrax protective antigen forms oligomers

(1990) Delineation of the functional site of a snake
venom cardiotoxin: preparation, structure, and function
of monoacetylated derivatives. Biochemistry 29, 6480–
6489.
23 Venema RC, Sayegh HS, Arnal JF & Harrison DG
(1995) Role of the enzyme calmodulin-binding domain
in membrane association and phospholipid inhibition of
endothelial nitric oxide synthase. J Biol Chem 270,
14705–14711.
24 Lin SC, Lo YC, Lin JY & Liaw YC (2004) Crystal
structures and electron micrographs of fungal volva-
toxin A2. J Mol Biol 343, 477–491.
25 Eisenberg D, Weiss RM & Terwilliger TC (1982) The
helical hydrophobic moment: a measure of the amphi-
philicity of a helix. Nature 299, 371–374.
26 Hsu YT & Youle RJ (1997) Nonionic detergents induce
dimerization among members of the Bcl-2 family. J Biol
Chem 272, 13829–13834.
27 Bhakdi S, Fussle R & Tranum-Jensen J (1981) Staphy-
lococcal alpha-toxin: oligomerization of hydrophilic
monomers to form amphiphilic hexamers induced
through contact with deoxycholate detergent micelles.
Proc Natl Acad Sci USA 78, 5475–5479.
28 Ole JB, James HG, Thorkild CB-H & Jesper BH (1982)
Electroimmunochemical analysis of amphiphilic proteins
and glycolipids stained with Sudan Black-containing
detergent micelles. Electrophoresis 3, 89–98.
29 Tone B & Ole JB (1986) Electrophoretic migration velo-
city of amphiphilic proteins increases with decreasing
Triton X-100 concentration: a new characteristic for

A, Bugge TH & Leppla SH (2005) Intermolecular com-
plementation achieves high-specificity tumor targeting
by anthrax toxin. Nat Biotechnol 23, 725–730.
39 Rono B, Romer J, Liu S, Bugge TH, Leppla SH &
Kristjansen PE (2006) Antitumor efficacy of a urokinase
activation-dependent anthrax toxin. Mol Cancer Ther 5,
89–96.
40 Kaku H, Tanaka Y, Tazaki K, Minami E, Mizuno H &
Shibuya N (1996) Sialylated oligosaccharide-specific
plant lectin from Japanese elderberry (Sambucus sie-
boldiana) bark tissue has a homologous structure to type
II ribosome-inactivating proteins, ricin and abrin.
cDNA cloning and molecular modeling study. J Biol
Chem 271, 1480–1485.
41 Wu YH, Shih SF & Lin JY (2004) Ricin triggers apop-
totic morphological changes through caspase-3 cleavage
of BAT3. J Biol Chem 279, 19264–19275.
42 Shih SF, Wu YH, Hung CH, Yang HY & Lin JY (2001)
Abrin triggers cell death by inactivating a thiol-specific
antioxidant protein. J Biol Chem 276, 21870–21877.
43 Wilchek M, Miron T & Kohn J (1984) Affinity chroma-
tography. Methods Enzymol 104, 3–55.
Supplementary material
The following supplementary material is available
online:
Table S1. Sequences of the primers used for cloning
volvatoxin A1 (VVA1) cDNA and peptide competi-
tors.
Fig. S1. Nucleotide and deduced protein sequence of
volvatoxin A1 (VVA1). The nucleotide sequence and

tion products were analyzed by 10% SDS ⁄ PAGE.
Fig. S4. Anti-volvatoxin A2 (VVA2) IgG. VVA2 and
volvatoxin A1 (VVA1) were analyzed by 10%
SDS ⁄ PAGE, followed by western blots and anti-VVA2
IgG.
This material is available as part of the online article
from
P T. Wu et al. VVA1 is a novel toxin regulator of VVA2
FEBS Journal 273 (2006) 3160–3171 ª 2006 The Authors Journal compilation ª 2006 FEBS 3171