Developmental and Comparative Immunology 48 (2015) 164–170

Contents lists available at ScienceDirect

Developmental and Comparative Immunology

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / d c i

Molecular cloning and expression analysis of the Ajuba gene of grass

carp (Ctenopharyngodon idella) involved in cellular response to
viral infection
Yanan Zhang, Hao Wang, Yan Li, Dan Xu, Liqun Lu *
Key Laboratory of Freshwater Fishery Germplasm Resources, Ministry of Agriculture of P. R. China, Shanghai Ocean University, Shanghai 201306, China

A R T I C L E I N F O A B S T R A C T

Article history: Ajuba belongs to the LIM domain proteins, which are involved in the assembly of the extracellular matrix
Received 4 August 2014 and, along with associated proteins, regulate target genes that connect the extracellular matrix and the
Revised 6 October 2014 cytoskeleton. In the present study, we characterized the entire cDNA sequence of the Ajuba gene from
Accepted 6 October 2014
grass carp (gcAjuba). The gcAjuba cDNA contained an open reading frame (ORF) of 2121 bp encoding a
Available online 16 October 2014
polypeptide of 706 amino acids with an estimated molecular mass of 75.966 kDa and three LIM domains
in the C-terminal. The transcriptional level of gcAjuba was significantly up-regulated following the stim-
Keywords:
ulation of virus in vitro. Sub-cellular location of gcAjuba and GCRV-JX01 NS26 proteins did not overlap
Ajuba
Grass carp in the cytoplasm and no direct interaction between gcAjuba and the protein NS26 was detected by co-
GCRV immunoprecipitation (CO-IP) test in grass carp kidney cells. Based on these results, the gcAjuba is determined
Inducible gene to be an immediately inducible gene responding to viral infection and in vivo association of gcAjuba with
NS26 could not be confirmed, which has been suggested by yeast two-hybrid assay in previous report.
© 2014 Elsevier Ltd. All rights reserved.

1. Introduction
LIM domain proteins are involved with protein–protein inter-
actions (Dawid et al., 1998) and the LIM domain is rich in conserved
cysteine and histidine residues that enable a stable tertiary struc-
ture when combined with Zn2+ (Ostendorff et al., 2002). Mammalian
LIM domain proteins are categorized into four groups (Bach, 2000;
Gill, 1995; Zheng and Zhao, 2007). The first group consists of two
tandem N-terminal LIM domains and one transcription activation
domain that are mainly involved in cell differentiation and cell fate;
the second group consists of two or more LIM domains clustered
at the N- or C-termini and associated with regulating cytoskel-
eton remodelling. The third group consists of three or four LIM
domains at the C-termini and different N-terminal domains whose
role is to bind with cytoskeletal protein in cell adhesion processes
(Marie et al., 2003; Pratt et al., 2005). The fourth group, similar to
group three in structure, participates in cell cycle regulation and
actin polymerization (Zheng and Zhao, 2007).
The Zyxin/Ajuba family of cytosolic LIM domain-containing pro-
teins (Ajuba, TRIP6, LPP, LIMD1, WTIP, Zyxin, Migfillin) is the third
group (Petit et al., 2005). These proteins have the potential to shuttle
* Corresponding author. Key Laboratory of Freshwater Fishery Germplasm
Resources, Ministry of Agriculture of P. R. China, Shanghai Ocean University, Shanghai
201306, China. Tel.: +86 2161900453; fax: +86 2161900454.
E-mail address: lqlv@shou.edu.cn (L. Lu).
from sites of cell adhesion into the nucleus and can be candidate
transducers of environmental signals (Feng and Longmore, 2005).
Among them, Ajuba consists of three LIM domains at the C-terminal
and is located in the cytoplasm where it interacts with a variety of
other proteins (Wang and Gilmore, 2003). Studies on the func-
tions of Ajuba have to date been concentrated in mammals. Ajuba
is a component of the IL-1 signalling pathway modulating IL-1-
induced NF-κB activation by influencing the assembly and activity
of the aPKC/p62/TRAF6 multiprotein signalling complex (Feng and
Longmore, 2005). NF-κB is one of the best-characterized transcrip-
tion factors. It is expressed ubiquitously and regulates the expression
of many genes, most of which encode proteins that play an impor-
tant and often determining role in the processes of immunity and
inflammation (O’Neill and Kaltschmidt, 1997). So, Ajuba is be-
lieved to be involved in the immunity system by NF-κB signal
transduction pathway.
Ajuba also contributes to the formation or strengthening of cell–
cell adhesion and regulates cell growth and differentiation decisions
(Feng and Longmore, 2005; Kanungo et al., 2000). Ajuba partici-
pates in the assembly of the extracellular matrix and regulates target
genes involved in the connection process of the extracellular matrix
and the cytoskeleton (Hou et al., 2008; Kisseleva et al., 2005). Ajuba
also plays an important role in some diseases. For example, Ajuba
can bind and activate the Aurora-A protein that is associated with
tumour development (Hirota et al., 2003).
Grass carp (Ctenopharyngodon idella) is an important commer-
cial fish that is widely grown in Eastern Asia (FAO, 2010). Grass carp

http://dx.doi.org/10.1016/j.dci.2014.10.002
0145-305X/© 2014 Elsevier Ltd. All rights reserved.
 Y. Zhang et al./Developmental and Comparative Immunology 48 (2015) 164–170
haemorrhagic disease, caused by the Grass carp reovirus (GCRV),
is a major problem for grass carp aquaculture (Chen et al., 2013).
GCRV has been assigned to the genus Aquareovirus in the family
reoviridae (Fauquet et al., 2005) and consists of 11 dsRNA genomic
fragments, which encode seven structural proteins (VP1–VP7) and
five non-structural proteins (NS16, 26, 31, 38 and 80) (Zhang et al.,
2010). It remained a puzzle for the NS26 protein that had no ho-
mologue in Mammalian reovirus or any other protein in available
database (Attoui et al., 2002). In a study to screen interacting cel-
lular proteins with grass carp reovirus using yeast two-hybrid system,
we identified that the GCRV-JX01 NS26 protein might interact with
three proteins: gcAjuba, CiLITAF and LOC571286 (Wang et al., 2013a).
Recent evidence indicated that NS26 could participate in cell–cell
fusion through cooperation with NS16 in aquareovirus infection (Guo
et al., 2013). Since the mammalian Ajuba family of LIM proteins
are adaptor proteins within multi-protein complexes that connect
cell–cell and cell matrix contact proteins to the cytoskeleton (Kim
et al., 2012), we are interested to characterize the grass carp Ajuba
gene and its possible involvement in cellular response to virus
infection.
In this report, the coding sequence of the entire cDNA of Ajuba
from grass carp (gcAjuba) was determined and analysed, in vivo as-
sociation of gcAjuba with GCRV NS26 was investigated, and
expression pattern of gcAjuba in response to GCRV infection was
characterized.
2. Materials and methods
2.1. Molecular cloning of the entire cDNA of gcAjuba
Grass carp kidney (CIK) commercial cells used in this study were
obtained from China Center for Type Culture Collection and grown
in 25 cm culture flask in the medium M199 supplemented with 10%
inactivated foetal calf serum (Gibco BRL) prior to being incubated
at 28 °C. To avoid the loss of the essential genes, the passage times
of original CIK cell lines were limited to 3–5 in our experiments.
The total RNA was extracted from 5 × 106 CIK cells using 2 mL TRIzol
Reagent (Invitrogen, USA) and reverse-transcribed into cDNA using
the SMARTer™ RACE cDNA Amplification Kit (Clontech, USA) ac-
cording to the manufacturer’s protocol. The primary PCR primers
were designed according to sequences obtained from a previous
study (Ajuba-F1 and Ajuba-R1, Supplementary Table S1). The primary
PCR was set up using cDNA generated from the total RNA of CIK cells
as the template. The PCR product was purified using the Wizard SV
Gel and PCR Clean-Up System (Promega, USA), then cloned into a
pMD19-T Vector (TaKaRa, Japan) and transformed into DH5α
competent cells.
To obtain the full-length cDNA sequence of gcAjuba, 3′ and 5′
rapid amplification of cDNA ends (RACE) was carried out using the
SMARTer™ RACE cDNA Amplification Kit (Clontech). The 3′ and 5′
RACE were performed as previously described (Shen et al., 2014),
but using total cellular RNA as the amplification template. Briefly,
3′RACE and 5′RACE were performed using gene-specific primers
(gcAjuba-R2 and gcAjuba-F3, Supplementary Table S1) and adapter
primers (UPM and NUP, Supplementary Table S1). The full se-
quence of gcAjuba was confirmed with the primers gcAjuba-F4/
gcAjuba-R4 and gcAjuba-F5/gcAjuba-R5 (Supplementary Table S1).
All amplification products were cloned and sequenced.
2.2. Bioinformatics analysis of gcAjuba gene
The BLAST program from the National Center for Biotechnol-
ogy Information (NCBI) was used to search homologous sequences
in GenBank (http://blast.ncbi.nlm.nih.gov). ORF Finder (http://
www.ncbi.nlm.nih.gov/projects/gorf/) was used to predict the open
reading frame (ORF) and deduced protein sequence of gcAjuba. The
165
ProtParam tool at ExPASy (http://web.expasy.org/protparam/)
was used to analyse the deduced amino acid sequence. Multiple
sequence alignments were generated with DNAMAN software.
Phylogenetic analysis of the amino acid sequences was performed
using the Neighbour-Joining (NJ) algorithm in MEGA version
5.0.
2.3. Virus preparation and infection
GCRV-JX01 were isolated from diseased grass carps with typical
haemorrhagic symptoms for the first time and maintained in the
laboratory (Wang et al., 2013b). The 90% confluent CIK monolayer
cells in five bottles of 100 cm culture flasks were infected with GCRV-
JX01. Culture supernatant was collected after the death of 90% of
infected cells. In brief, the virus particles were purified from the su-
pernatant of infected CIK cells for the observation of CPE by
ultracentrifugation (Fang et al., 2008). Virus titration was per-
formed via the standard TCID50 (50% tissue culture infective dose)
assay (He et al., 2011). CIK cells were maintained as stock cultures
in DMEM and re-plated 2 days before infection with GCRV-JX01. Cells
were infected for 1 h, and fed with 200 μL fresh medium. At 48 h
post infection, 96-well plates were observed under light micro-
scope for typical CPE. The TCID50 value was calculated using the
Reed–Muench method.
For virus infection, CIK cells cultured in six-well cell culture plates
were infected with GCRV-JX01 at a multiplicity of infection (MOI)
value of 1.0. Controls were left uninfected in the experiment. Three
repeats (three wells) were performed in experimental groups and
control groups. After allowing 2 h for absorption, all unattached
viruses were removed and the infected cells were continuously cul-
tured with the M199 medium supplemented with 10% FBS. Cells
were collected at 0, 4, 8, 12, 24 h p.i.
2.4. RNA extraction and quantitative RT-PCR
Total RNA was isolated from both the infected cells and the control
cells using TRIzol (Invitrogen) and then subjected to DNase I treat-
ment (Promega) according to the manufacturer’s protocol for
real-time PCR analysis (RT-PCR). RNA was assayed by a Nanodrop
2000 spectrophotometer. Approximately 200 ng of RNA from each
sample was reverse-transcribed using PrimerScript First Strand cDNA
Synthesis Kit (Takara). The first-strand cDNA was subsequently used
as the template for the RT-PCR. The grass carp elongation factor 1a
(EF1a) gene was amplified as an internal standard reference gene
(Su et al., 2011). The specific primers RT-gcAjuba-F6/R6 (Supple-
mentary Table S1) used for quantitative RT-PCR (qRT-PCR) displayed
a single peak in melting curve analysis with an amplification
efficiency close to the theoretical 100%. RT-PCR was carried out in
a 20 μL reaction volume containing 5 μL of 1:10 diluted original
cDNA, 10 μL of 2 × SYBR Green Master Mix (Bio-Rad, USA), 2 μL of
each primer (20 pmol/μL), and 3 μL of PCR grade water using the
Bio-Rad CFX 96 Real-time PCR machine. The triplicate fluores-
cence intensities of each sample, as measured by crossing-point (Ct)
values, were compared and converted to fold differences by the rel-
ative quantification method (Niu et al., 2014). The gcAjuba
transcriptional level was calculated by 2−ΔΔCT method. The data were
subjected to analysis of Student’s t-test and the P values less than
0.05 (P < 0.05) were considered statistically significant.
2.5. Recombinant plasmid construction and sub-cellular localization
For the construction of recombinant plasmids pEGFP-gcAjuba and
pcDNA3.1-NS26, primers pEGFP-gcAjuba-F/R, containing XhoI and
BamHI sites (Supplementary Table S1), were used to amplify the ORF
of gcAjuba. The PCR products were purified and cloned into a pEGFP-
N1 expression vector to get pEGFP-gcAjuba. Similarly, primers
166 Y. Zhang et al./Developmental and Comparative Immunology 48 (2015) 164–170
pcDNA-NS26-F/R, containing HindIII and EcoRI sites (Supplemen-
tary Table S1), were used to amplify the ORF of NS26. The PCR
products were purified and cloned into a pcDNA™3.1/V5-His ex-
pression vector to get pcDNA3.1-NS26. The constructed recombinant
expressing plasmid was extracted, purified using a NucleoSpin
plasmid quick-pure kit (Macherey-Nagel, USA) according to the
manufacturer’s protocol and confirmed by sequencing.
To determine the location of the protein gcAjuba and NS26 in
cells, CIK cells were seeded into six-well cell culture plates for about
18–20 h (with an 80%–90% confluent monolayer) using M199
medium containing 10% of the inactivated foetal calf serum to
transfect. About 2 × 106 CIK cells were co-transfected with 2 μg
pEGFP-gcAjuba and 2 μg pcDNA3.1-NS26 using Lipofectamine 2000
(Invitrogen) following the manufacturer’s protocol. After 20 h,
immune-fluorescence assay (IFA) was used to observe the ex-
pressed position of NS26 protein (Terry et al., 2011; Zhou et al., 2014).
Cells were fixed in methanol, then incubated with His-Tag mouse
mAb (EarthOx, USA) diluted 1:500, and stained with the Texas Red
AffiniPure Goat anti-mouse IgG (EarthOx) diluted 1:100. Nuclei were
stained with DAPI and observed by a fluorescence microscope. The
CIK cells co-transfect with plasmid pEGFP-gcAjuba and pcDNA3.1-
NS26 were subjected to Western blotting analysis after 20 h. The
primary anti-GFP Tag and anti-His Tag antibody (Abmart, China)
was diluted 1:5000. The secondary Goat Anti-Mouse IgG-HRP
(Abmart) antibody was diluted 1:6000.
2.6. Co-immunoprecipitation (CO-IP)
To verify the possible interaction between gcAjuba and GCRV
NS26, CO-IP was performed on incubated CIK cells kept in six-
well culture plates. About 1.5 × 107 CIK cells were co-transfected with
12 μg pEGFP-gcAjuba and 12 μg pcDNA3.1-NS26 using Lipofectamine
2000 (Invitrogen). Plasmids pcDNA3.1-NS26 and pEGFP-N1 were co-
transfected into CIK cells to serve as controls, as well as pcDNA™3.1/
V5-His and pEGFP-gcAjuba. Twenty-four hours later, cells were
scraped, washed with PBS, and lysed in a RIPA buffer (Lettau et al.,
2014; Lutz et al., 2012). Cellular debris was removed by centrifu-
gation. Primary antibody (anti-GFP, Abmart) and Protein A/G
PLUS-Agarose (Santa Cruz Biotechnology, Inc.) was added to the
supernatant according to the operation manual. The precipitate of
Protein A/G PLUS-Agarose and supernatant were collected for
western blot analysis. The primary His-Tag mAb (EarthOx),
GFP-Tag mouse mAb, GFP-Tag rabbit mAb was diluted 1:5000
respectively. The secondary Goat Anti-Mouse/Rabbit IgG-HRP
(Abmart) antibody was diluted 1:6000 respectively.
3. Results
3.1. Cloning and sequence characterization analysis
The full-length cDNA of gcAjuba (GenBank accession number:
KC551289.1) was isolated from the CIK cells by RACE and se-
quenced successfully. The nucleotide sequence of gcAjuba cDNA was
3980 bp in length, including a 5′-untranslated region (5′UTR) of
1215 bp, a 3′-untranslated region (3′UTR) of 644 bp and a 2121-
bp ORF encoding a precursor of 706 amino acids. The ProtParam
results showed that its molecular weight was 75.966 kDa, its the-
oretical pI was 7.87 and the instability index (II) was 64.48, indicating
that it is an unstable protein. Using the NCBI database, we identi-
fied three LIM domains that had a Zn2+ binding site in the C-terminal
of gcAjuba coding protein.
3.2. Phylogenetic analysis
BLAST results showed the amino acid sequence comparison of
gcAjuba and its homologues from other species. The gcAjuba coding
sequence demonstrated a high degree of amino acid identity with
Zebra fish (~87%), the Bicolor damselfish (64%), Japanese medaka
(61%), and around 53% similarity to Ajuba from many other species
(human, cattle and house mouse). In general, the amino acid se-
quence of the gcAjuba protein from these species exhibited relatively
high homology (Fig. 1). Phylogenetic analysis showed that the
gcAjuba clustered most closely with the Ajuba protein of other fish
species (particularly that of Zebra fish) and was furthest phyloge-
netically from mammalian Ajuba (Fig. 2). These results indicate that
the gene we isolated is likely to be a duplicate of the Ajuba protein
from grass carp. The accession numbers of all the species are listed
in Supplementary Table S2.
3.3. Quantitative RT-PCR of gcAjuba in response to virus infection
To demonstrate whether GCRV-JX01 regulates the expression of
gcAjuba, we examined the gcAjuba transcriptional level after the virus
infection by qRT-PCR. As shown in Fig. 3, we found that gcAjuba
mRNA expression was up-regulated after infection and reached a
peak level at 4 h (3.1-fold, P < 0.05), after that the expression levels
declined to a lower level at 8 h (1.5-fold, P > 0.05), 12 h (1.1-fold,
P > 0.05), and 24 h (1.4-fold, P > 0.05).
3.4. Co-localization of gcAjuba with GCRV NS26
If NS26 and gcAjuba interacted as yeast two-hybrid assay sug-
gested (Wang et al., 2013a), both of them should display similar
localization in CIK cells. To investigate the sub-cellular location of
the gcAjuba and NS26 proteins, we co-transfected CIK cells with re-
combinant plasmids pEGFP-gcAjuba and pcDNA3.1-NS26. After 20 h,
IFA was used to observe the expressed position of NS26 protein. Then
the CIK cells were stained with DAPI and observed by fluores-
cence microscope. As shown in Fig. 4A, the His-fused NS26 protein
(red) was expressed ubiquitously and the EGFP-fused gcAjuba protein
(green) was patchily distributed in the cytoplasm. This indicated
that both gcAjuba and NS26 were expressed in the cytoplasm, but
their locations did not overlap. To further verify gcAjuba protein and
NS26 protein expression, western blot analysis was conducted using
the pEGFP-Ajuba and pcDNA3.1-NS26 transfected cell samples.
Results indicated that the gcAjuba and NS26 protein was indeed ex-
pressed in CIK cells (Fig. 4B). The co-localization test did not support
the idea that NS26 holds the potential to interact with gcAjuba.
3.5. Co-immunoprecipitation (CO-IP)
To further test the possible interaction of gcAjuba with GCRV
NS26, CO-IP was performed on CIK cells expressing both His-
NS26 and GFP-gcAjuba. We found that fusion protein GFP-Ajuba
and His-NS26 were both expressed after cotransfection with pEGFP-
Ajuba and pcDNA3.1-NS26, as reflected by their existence in the
CO-IP supernatant (Fig. 5A). However, we failed to detect His-
NS26 when anti-GFP was used to precipitate GFP-Ajuba, or detect
GFP-Ajuba when anti-His was used to precipitate His-NS26 (Fig. 5B).
Thus, CO-IP test failed to co-precipitate either of the two proteins,
which validated the above co-localization test.
4. Discussion
Ajuba is an important cytosolic LIM protein that shuttles into the
nucleus and affects cell proliferation and the fate of cells (Kanungo
et al., 2000). However, there have been few reports about the role
of Ajuba in fish until now. Previous work, in a study to screen in-
teracting cellular proteins with grass carp reovirus using yeast two-
hybrid system, gcAjuba was identified to bind the NS26 protein
encoded by the S11 genomic fragment of GCRV JX01 (Wang et al.,
2013a). Moreover, Ajuba participates in the activation of NF-κB, a
 Y. Zhang et al./Developmental and Comparative Immunology 48 (2015) 164–170 167

Fig. 1. Amino acid sequence comparison of the gcAjuba protein with its homologues from other species using the DNAman software. GenBank accession numbers are as
detailed in Supplementary Table S2.
168 Y. Zhang et al./Developmental and Comparative Immunology 48 (2015) 164–170
Fig. 2. Neighbour-joining phylogenetic tree generated for Ajuba amino acid sequences with its homologues from other species using the Mega 5.0 program. Robustness
was ensured by 1000 bootstrap replications. GenBank accession numbers are as detailed in Supplementary Table S2.
transcription factor in immunity system (Feng and Longmore, 2005;
Rahman et al., 2002). These reports drove us to investigate the in-
volvement of gcAjuba in anti-viral cellular response.
Phylogenetic analyses are central to many research areas in
biology and typically involve the identification of homologous se-
quences (Dereeper et al., 2008). In this study, we successfully
identified and characterized the Ajuba gene of grass carp from CIK
Fig. 3. Expression profile of gcAjuba in CIK cells after infection with GCRV. The rel-
ative expression of the transcript from qRT-PCR was expressed as fold change over
control samples and normalized to the EF1a control. P < 0.05 was considered
statistically significant.
cells. The amino acid sequence of gcAjuba shared 87% identity with
that of zebra fish and was homologous to mammal Ajuba by mul-
tiple sequence alignments and phylogenetic analysis. We found that
the gcAjuba protein contains three LIM domains that have a Zn2+
binding site in the C-terminal, which is consistent with the Ajuba
LIM proteins of the Zyxin family and similar with Ajuba protein of
mammals (Goyal et al., 1999; Jamesa et al., 2010).
To characterize the expression pattern of gcAjuba after virus in-
fection, we monitored gcAjuba transcriptional level at 0, 4, 8, 12,
24 h p.i. The expression of gcAjuba gene was significantly up-
regulated after infection. The maximum expression level was
observed at 4 h p.i., which suggested an immediate response to virus
infection. GCRV has been assigned to a newly established
Aquareovirus genus in the family of Reoviridae (Ma et al., 2011). Also,
aquareoviruses can induce cell–cell fusion and multinucleated syn-
cytium formation (Racine et al., 2009). Ajuba, as a new component
at cadherin-mediated cell–cell junctions, should participate in the
formation of cellular adhesive complexes by Ajuba/Zyxin family of
LIM proteins (Marie et al., 2003; Pratt et al., 2005). The mammali-
an Ajuba LIM proteins (Ajuba, LIMD1, WTIP) are cytosolic adapter
proteins that exhibit the potential to communicate cell adhesive
events with nuclear responses to remodel epithelia (Das Thakur et al.,
2010). Therefore, we hypothesized that the up-regulated expres-
sion of gcAjuba may contribute to the morphology change of
intercellular junctions after GCRV JX01 infection.
The experiment of sub-cellular location indicated that the gcAjuba
protein was expressed and patchily distributed in the cytoplasm.
Ajuba has been reported to be a co-repressor for the SNAG domain,
which is present in a variety of proto-oncogenic transcription factors
(Ayyanathan et al., 2007; Chiang and Ayyanathan, 2013). Similar-
ly, Ajuba functions as a histone deacetylase-dependent co-repressor
for auto-regulation of the growth factor-independent-1 transcrip-
tion factor (Montoya-Durango et al., 2008) and plays a central role
in regulating the SHF during heart development by linking RA sig-
nalling to the function of Isl1, a key transcription factor in cardiac
progenitor cells (Witzel et al., 2012). But NS26 obviously did not
co-localize with gcAjuba in our co-localization assay. Further-
more, CO-IP experiment failed to indicate the interaction of
 Y. Zhang et al./Developmental and Comparative Immunology 48 (2015) 164–170 169

Fig. 4. Localization test of GFP-Ajuba and His-NS26 in CIK cells. Cells were co-transfected with the plasmid pEGFP-gcAjuba and pcDNA3.1-NS26. (A) Sub-cellular localiza-
tion of recombinant gcAjuba (green) and NS26 (red). Sub-cellular localization of NS26 was performed using IFA with Anti-His Tag Monoclonal Antibody and Texas Red AffiniPure
Goat Anti-Mouse IgG(H + L) (EarthOx). The cells were stained with DAPI and examined under fluorescent microscope. (B) Verification of the transient expression of recom-
binant plasmid pEGFP-N1-gcAjuba and pcDNA3.1-NS26 by Western blotting. Cells were detected with GFP-Tag mAb (Abmart) and His-Tag mAb (Abmart), respectively. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

over-expressed protein gcAjuba and NS26 in CIK cells. An
obviousexplanation may be that there is no direct interaction
between gcAjuba and NS26. But co-localization test did indicate that
both gcAjuba and GCRV-JX01 NS26 proteins were expressed mainly
in the cytoplasm, and it is still possible that these two proteins could
interact with each other under certain conditions. Since Ajuba modu-
lated Rac dynamics at contact depending on its phosphorylation
status (Nola et al., 2011), and induced the autophosphorylation and
consequent activation of Aurora-A (Hirota et al., 2003), we specu-
lated that the phosphorylation, methylation or proper folding status
of gcAjuba may affect its interaction with NS26. A third partner might
exist to stimulate the interaction. Thus, it remained to be resolved
whether gcAjuba did interact directly with NS26 under certain
conditions.
In conclusion, this study characterized the full-length se-
quence of cDNA of gcAjuba. The sequence data and details of the
genomic structure of the gcAjuba gene provided critical informa-
tion for further studies investigating the function of this gene in grass
carp. CO-IP experiment and co-localization assay of gcAjuba and
GCRV NS26 indicated that the two proteins, unlike in yeast, were
not associated with each other in grass carp cells. However, GCRV-
JX01 infection caused significant up-regulation of gcAjuba at 4 h,
indicating that gcAjuba is an immediately inducible gene respond-
ing to viral infection.
Acknowledgements
This work was supported by grants from the National Natural
Science Foundation of China (No. 31372561) and the Earmarked Fund
for China Agriculture Research System (No. CARS-46-12). The authors
thank the support from Shanghai Universities First-class Disci-
plines Project of Fisheries.

Appendix: Supplementary Material

Supplementary data to this article can be found online at

doi:10.1016/j.dci.2014.10.002.

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