Structural and functional studies of STAT1 from Atlantic salmon (Salmo salar)
© Skjesol et al; licensee BioMed Central Ltd. 2010
Received: 30 September 2009
Accepted: 30 March 2010
Published: 30 March 2010
Type I and type II interferons (IFNs) exert their effects mainly through the JAK/STAT pathway, which is presently best described in mammals. STAT1 is involved in signaling pathways induced by both types of IFNs. It has a domain-like structure including an amino-terminus that stabilizes interaction between STAT dimers in a promoter-binding situation, a coiled coil domain facilitating interactions to other proteins, a central DNA-binding domain, a SH2 domain responsible for dimerization of phosphorylated STATs and conserved phosphorylation sites within the carboxy terminus. The latter is also the transcriptional activation domain.
A salmon (Salmo salar) STAT1 homologue, named ssSTAT1a, has been identified and was shown to be ubiquitously expressed in various cells and tissues. The ssSTAT1a had a domain-like structure with functional motifs that are similar to higher vertebrates. Endogenous STAT1 was shown to be phosphorylated at tyrosine residues both in salmon leukocytes and in TO cells treated with recombinant type I and type II IFNs. Also ectopically expressed ssSTAT1 was phosphorylated in salmon cells upon in vitro stimulation by the IFNs, confirming that the cloned gene was recognized by upstream tyrosine kinases. Treatment with IFNs led to nuclear translocation of STAT1 within one hour. The ability of salmon STAT1 to dimerize was also shown.
The structural and functional properties of salmon STAT1 resemble the properties of mammalian STAT1.
Interferons (IFNs) are cytokines that play a major role in host defense against viral pathogens [1, 2]. Mammalian type I IFNs (IFNα/β) are produced by many cell types and confer antiviral activities on them, while type II IFN (IFNγ) is produced mainly by T lymphocytes and natural killer cells when stimulated by macrophage derived cytokines. IFNγ elicits broad effects, particularly on cells of the immune system. The transmission of both type I IFNs and IFNγ signals are dependent on the activation of the transcription factor STAT1 (signal transducer and activator of transcription). STAT family proteins are critical to the action of most cytokines and growth factors, as they are latent cytoplasmic transcription factors that directly activate signaling pathways upon being phosphorylated [3–5].
The activation of STAT is encompassed as part of evolutionary conserved pathways by which signals can be transduced from the membrane to the nucleus rapidly. The classical view is that type I IFN (IFNα/β) signals through STAT1/STAT2 heterodimers, while IFNγ signals through STAT1 homodimers [6, 7]. The binding of secreted type I IFNs to the two subunit receptor (IFNAR1/IFNAR2) results in activation of the Janus-activated kinase 1 (JAK1) and tyrosine kinase 2 (TYK2), which are associated with the cytoplasmic tail of IFNAR1/2. The signal is cascaded further by tyrosine phosphorylation of STAT1 and STAT2 [3, 8, 9]. The STATs heterodimerize and together with interferon regulatory factor 9 (IRF9) form a complex named ISGF3. This complex enters the nucleus where it associates with specific promoter elements (termed the IFN-stimulated response element or ISRE) to activate the transcription of IFN-stimulated genes (ISGs) . IFNγ signals through an IFNγ-specific receptor (IFNGR1/IFNGR2) to JAK1 and JAK2 resulting in tyrosine phosporylation and homodimerization of STAT1 . STAT1 homodimers enter the nucleus and bind the IFNγ-activation site (GAS) which is present in the promoter of certain ISGs [3, 11]. However, in addition to the phosphotyrosine SH2 domain interactions of the active forms of STATs, unphosphorylated STATs can form dimers of a different conformation through their N-terminal domain . Also, STAT1 can be found in both the cytoplasm and the nucleus without cytokine stimulation of cells .
Facilitated nuclear translocation of such large complexes requires the nuclear pore complex [5, 14]. STAT1 and STAT2 do not contain classical nuclear localization signals (NLS) which is normally necessary to be recognized by the importin receptor, but dimerization of STATs results in conformational changes that establish NLS activity [13, 15, 16]. After activation of their target genes, STATs are dephosphorylated, released from the DNA and shuttled back to the cytoplasm [12, 17, 18]. Consistent with the importance of this pathway in mediating the actions of IFNs, mice with no STAT1 have no innate response to either bacterial or viral infections as a result of dysfunctional IFN signaling . Moreover, a number of viruses have the capacity to block the activation of STAT1 by IFN to evade the defense from the host immune system .
Recently, significant progress has been made in identifying and characterizing fish genes related to the IFN system, including several type I IFN genes [21–23], IFNγ [24–28] and antiviral genes . Far less is known about the factors that are involved in IFN-signaling in fish, including the JAK-STAT pathway, although STAT1 homologs have been cloned from several fish species [29–31]. Additionally, a STAT2 gene was recently identified in salmon , and TYK2 and JAK1 have been cloned from green pufferfish (Tetraodon fluviatilis) [33, 34].
In the present work we describe the identification and characterization of a STAT1 gene from Atlantic salmon. To get insight into the role of STAT1 in response to cytokines and viruses in salmon we have studied the expression and activation of STAT1 in primary leukocyte cultures and in different salmonid cell-lines upon type I and type II IFN treatment and viral infections. The ability of STAT1 to be phosphorylated and to translocate to the nucleus is critical for its role as a transcription factor. By employing a salmon STAT1 antibody the localization of STAT1 in different cells in response to IFN-treatment were studied. Furthermore, STAT1 phosphorylation was detected using a phosphotyrosine specific antibody after treatment with the same stimulants. Such studies have not been performed in any teleost species earlier. Both IFN-a1 and IFNγ treatment led to tyrosine phosphorylation and STAT1 was also shown to be translocated to the nucleus after stimulation with IFN-a1 and IFNγ. We also show, using two different in vitro methods, that salmon STAT1 is able to form dimers.
Cloning of STAT1
Primer sequences and their applications in this study.
Sequence 5' - 3'
ssSTAT1 2204 fw
Isoform detection (PCR)
ssSTAT 1gap rev
Isoform detection (PCR)
The amino acid sequence of the ssSTAT1a clone contained several prototypic features and conserved domains crucial for STAT1 functions (Figure 1). In the most C-terminal part of the sequence, the transcriptional activation domain (TAD), a conserved serine phosphorylation site was found in position 719 (homologous to serine 727 in human STAT1). This phosphorylation site is known to be crucial for the activation of transcription . A conserved tyrosine residue was located in position 695 (701 in human). Phosphorylation of this tyrosine residue is crucial for activating the STAT molecule and thereby enabling it to interact with the phosphotyrosine binding pocket located in the Src homology 2 (SH2) domain of other STAT molecules . An analysis of this conserved domain (using NCBI CDD ) revealed that a phosphotyrosine binding pocket was located in SH2 of ssSTAT1a, where all four of the residues composing this feature was present, which of the conserved arginine at position 597 is believed to be the most crucial for forming H-bonds with phosphate oxygens of the phosphotyrosine side chain . In addition residues important for DNA-binding (DNAB), nuclear import (NLS) and export (NES) [15, 39–41] were found to be conserved between mammalian and teleost STAT1. Residues crucial for these functions are marked with asterisks in Figure 1.
Tissue distribution of STAT1 mRNA
Induction and expression of STAT1 protein in salmon primary cells
Transcription levels of salmon STAT1 mRNA in salmon HK leukocytes
STAT1 protein expression levels in TO and CHSE-214 cells upon IFN-treatment and viral infections
Subcellular localization of STAT1
Salmon STAT1 is phosphorylated upon IFN treatment
Salmon STAT1 homodimerisation
Interaction between ssSTAT1a and ssSTAT1a detected in the yeast two-hybrid system.
++++ (TDO) +++ (QDO)
++++ (TDO) ++++ (QDO)
IFN induced immune responses in which STATs are required are among the best understood signaling systems in mammals. Although a number of proteins involved in the JAK/STAT signaling pathway have been cloned from fish, less is known about their function and whether the signaling resembles mammalian systems.
We have here cloned a cDNA that corresponds to the salmon STAT1 gene. A clustalW alignment confirmed that the cloned sequence was a STAT1 homolog sharing extensive amino acid identity with other salmon STAT1 isoforms and trout STAT1 (Table 1 and Figure 1). The 2 274 nucleotides open reading frame of the cloned cDNA has a structural arrangement of functional motifs that is similar to mammalian STAT1 suggesting that the salmon cDNA encode a functional protein.
So far STAT1 has been found in multiple fish species, including pufferfish, zebrafish (Danio rerio), rainbow trout, Atlantic salmon and Japanese flounder (Paralichthys olivaceus). Previous expression data, along with data presented here has revealed that piscine STAT1 is widely expressed in many tissues [30, 31, 46–49]. However, data concerning functional activity in lower vertebrates, such as STAT1 phosporylation and cellular localization upon stimulation of cells, is scarce. The presented data demonstrates for the first time that a teleost STAT1 protein is being activated by IFNs. Salmon STAT1 was shown to be tyrosine phosphorylated upon IFN-a1 and IFNγ stimulation of leukocytes, and additionally in TO cells. Also, relocalization of STAT1 into the nucleus of leukocytes and TO cells was observed following IFNγ stimulation. Our data show a more evident response for IFNγ than for IFN-a1 when studying nuclear translocation of STAT1 by microscopy. This was also consistent with the levels of phosphorylated STAT1 observed upon the different IFN-stimulations where IFNγ consistently gave higher levels compared to IFN-a1. STAT1 is believed to be involved in both type I and type II IFN signaling and their distinct responses could be due to unequal concentrations or activity of the cytokines used, or be dependent on differences in the kinetics of forming the complexes that enter the nuclei. We also showed that ssSTAT1a is able to form homodimers which is thought to be a prerequisite for entering the nucleus due to lack of a functional nuclear localization signal in its monomeric form . In unstimulated cells STAT proteins can exist as stable unphosphorylated dimers or monomers which are also shuttled over the nuclear membrane [50–52] and are able to regulate gene expression in unconventional manners , consistent with the observed presence of STAT1 in nuclear extracts of unstimulated cells.
The TO cell line is derived from salmon HK and it consists of heterogeneous cell types . The CHSE-214 cells are embryo cells derived from Chinook salmon (Oncorhynchus tshawytscha) . Both these cell lines are widely used as experimental systems to study immune responses in salmon [47, 55–60]. According to our results, the expression of STAT1 protein seems to be up-regulated upon type I and type II IFN treatment in both the cell lines and also in primary HK leukocytes. While the presented Western data are not quantitative, we provide qPCR data showing that type I IFN induce STAT1 expression in HK leukocytes to a greater extent (10-fold) than type II IFN (3-fold), and also by different kinetics, where the response to type II IFN peaked at an earlier time-point. Unlike the HK leukocytes, the splenocytes, which are mostly lymphoid-like cells, did not show increased abundance of STAT1 upon IFN-stimulation. The up-regulation of salmon STAT1 transcripts after IFN-a1 stimulation of TO cells is reported by others [31, 47], additionally the type I IFN inducer poly (I:C) has been shown to induce STAT1 mRNA expression in RTG-cells .
Following encounter with viral pathogens, CHSE-214 and TO cells did not seem to boost the levels of STAT1 protein, but stayed relatively constant. Mx-protein was induced 24-48 h after infection with ISAV, but did not respond to IPNV infection, which is in compliance with results reported earlier [57, 61, 62]. The uniform expression patterns in salmon tissues and in different cell-types treated in various ways indicate that it is likely that STATs are present in the cytoplasm in most resting tissues, alert and ready to be activated upon cellular receptor signaling. The signal transduction and activation of ISGs may lead to a feedback loop that amplifies IFN-responses and induces STAT1 in a secondary manner, STAT1 itself being an ISG. This is shown in human cells-lines where IFNγ-induced IRF-1 in concert with CREB binding protein acts as key up-regulator of STAT1 mRNA transcription by binding to a combined IRF-E/GAS element in the STAT1 promoter . Mutual regulation of STAT1 and IRF-1 indicates an intracellular amplifying circuit in response to IFN.
The STAT1 complexes formed in response to type I IFN in salmonid cells are presumably, like in mammals, distinct from those formed in response to type II IFN. Masking of the epitope for the STAT1 antibody after responding to IFN1a is a plausible explanation to the inability to detect nuclear STAT1 by confocal microscopy examination upon IFN-a1 stimulation. Under the denaturing conditions of SDS-PAGE and Western blotting STAT1 was detected in the nucleus of TO cells after treatment with IFN-a1. Also a small portion of STAT1 was detected in nuclear extracts of unstimulated cells, which is consistent with observations of STAT1 as a constitutive transcriptional regulator in mammalian systems [13, 64, 65]. Unlike TO cells and primary leukocytes, no relocalization of STAT1 from the cytoplasm to the nucleus was found upon IFNγ treatment of CHSE-214 cells. This could be due to the apparent low levels of endogenously expressed STAT1 protein in these cells. However, the expression levels were evaluated by Western blotting and the low detectable levels may be due to a lower affinity of the STAT1 antibody to Chinook salmon STAT1. Nevertheless, the levels of STAT1 in CHSE-214 were induced by both types of IFN, but considerably later (24 h, Figure 6) than the time points used for the confocal microscopy examination. No nuclear extracts were made in order to examine STAT1 expression in these cells. The presence of the IFN receptors (IFNAR and IFNGR) in the different cell-lines is also uncertain although a putative IFNγ-receptor was recently identified in rainbow trout . The differences in response endorse the assumption that there are distinct proteins involved in signaling from type I and type II IFNs. The presence of a STAT2 gene in salmon was recently reported , and the first teleost importin alpha gene was recently cloned from red seabream (Pagrus major) , both findings adding to the assumption that IFN signaling in fish resembles that of mammals.
Unlike the (most common) action of human IFNγ [24, 68], IFNγ in salmonids up-regulates Mx expression [55, 69]. This might be a consequence of indirect stimulation, as fish IFNγ can activate type I IFN  (and own unpublished data). Interestingly, recombinant IFNγ activates an ISRE-containing reporter-construct in a dose dependent manner whereas constructs containing only GAS elements give no response to either type of IFNs as shown by Castro et al. . This finding suggests that cross-talk between IFN signaling pathways occurs in fish. Overlapping effects of type I and II IFNs are also described in mammals [71, 72] In the absence of exogenously administered IFNs, over-expression of ssSTAT1a by transfection in TO cells activated the Mx-promoter at a very moderate level (Additional file 2, Supplemental Figure S1). Furthermore over-expression of ssSTAT1a gave no additionally increased Mx-promoter activity upon type I or II IFN supplement. This is probably due to lack of stoichiometric balance of factors that activate, or interact with, the over-expressed STAT1 molecules. Similar results are observed in mammalian cells with a slightly different IFNγ-responsive reporter .
Naturally occurring truncated forms of STATs can act as competitors of functional STATs and inhibit transcriptional activation . The ssSTAT1a gene differs from two other salmon STAT1s published in GenBank. The presence of more than one STAT1 isotype in salmon implies distinct functions for the different STAT1s, possibly at the level of transcription activation as the main differences are located in the TAD. The presented ssSTAT1a is longer than EU016199 (ssSTAT1)  while shorter than BT045567. Our attempt to detect both ssSTAT1a and BT045567 transcripts in salmon primary cells failed, and only ssSTAT1a was detected independent of the type of treatment the cells were subjected to. Other cell-types, signals or cellular conditions might favor BT045567 expression.
We have cloned a salmon STAT1 isoform possessing conserved structural domains. Expressional and functional studies of this gene suggest that salmon use the specialized JAK/STAT pathway for cytokine signaling as STAT1 is tyrosine phosphorylated and translocates to the nucleus upon IFN stimulation. Further details related to JAK/STAT signaling and viral infections of this economically important fish needs to be elucidated. The ability of IPNV to impair STAT1 activation/phosphorylation is currently being examined, and the interacting ability of activated STAT1 needs to be addressed.
For in vitro cell-culture studies non-vaccinated Atlantic salmon, Salmo salar L., strain Aquagen standard (Aquagen, Kyrksæterøra, Norway), 500-1000 g, was obtained from Tromsø Aquaculture Research Station (Tromsø, Norway). The fish were kept at 6 to 12°C in tanks supplied with running filtered sea water, and were fed according to appetite on commercial, dry food.
For tissues expression studies, non-vaccinated Atlantic salmon (~30 g), were obtained from SalmoBreed (Norway). The fish were kept in 150 l fresh water at 10 to 13°C, with an oxygen saturation >65% and were fed according to appetite on commercial, dry food. The fish were negative for the presence of infectious pancreatic necrosis virus and salmon alphavirus when screened by qPCR prior to the experiment.
The experiment was approved by the National Committee of Ethics as required by Norwegian law. Proper anaesthetics have been used and the number of fish was kept as low as possible to still get statistically significant results.
Cell cultures and virus
Atlantic salmon head kidney (HK) or spleen leukocytes were isolated as described by Jørgensen et al. . The density of the leukocyte suspensions was adjusted to 7 × 106 cells/ml. One ml of HK leukocytes was plated per well in 24-well plates in L-15 medium with 5% FBS, whereas one ml splenocytes were plated in RPMI-1640 with 5% FBS. After approximately 24 h of incubation at 14°C, the cells were washed with culture medium prior to stimulation.
Chinook salmon embryo cells (CHSE-214)  were grown as monolayers at 20°C, 5.0% CO2 in Eagle minimal essential medium with GlutaMAX (EMEM+GlutaMAX, Invitrogen) supplemented with 100 μg/ml streptomycin, 60 μg/ml penicillin, 1% non essential amino acids and 8% fetal bovine serum (FBS, Euroclone). For infection experiments and Western analyses CHSE-214 cells were seeded into 24-well plates (2 × 105cells/well) and grown to 80% confluence prior to infection.
TO cells originating from Atlantic salmon head kidney  were grown as monolayers at 20°C, 5.0% CO2 in Eagle minimal essential medium with GlutaMAX (EMEM+GlutaMAX, Invitrogen) supplemented with 100 μg/ml streptomycin, 60 μg/ml penicillin, 1% non essential amino acids and 5% fetal bovine serum (FBS, Euroclone).
HEK-293 cells (GP-293; Clontech) were maintained at 37°C, 5.0% CO2 in EMEM supplemented with 100 μg/ml streptomycin, 60 μg/ml penicillin, 4 mM L-glutamine and 10% fetal bovine serum (FBS).
Infectious pancreatic necrosis virus (IPNV) of the N1 strain, serotype Sp, was used in this study. The experiments were performed with a multiplicity of infection (MOI) of 4 infectious particles in CHSE-214 or TO cells. After absorption of the virus for 3-4 h in serum free culture medium, the medium containing virus was carefully removed from the cells. The infection was then carried out at 17.5°C in the presence of 2% FBS and cells harvested at different time points. Propagation and titration of virus was performed as described in Pedersen et al .
Infectious salmon anemia virus (ISAV) of the Norwegian reference strain Glesvaer 2/90  (isolate ISAV4, hemagglutinin GenBank accession number AF220607) was kindly provided by Dr. B. Annexing, National veterinary institute, Oslo, Norway, and used to infect TO cells at a MOI of 4. The infection was carried out at 17.5°C in the presence of EMEM with 2% FBS and the cells harvested at different time points.
Stimulation of cells
Recombinant Atlantic salmon IFN-a1 (previously named IFN-α1) was produced in HEK293 cells as described elsewhere . The salmon IFN-a1 used in this study had a titer of 24 237 U/ml as estimated by the formula given by Renault et al.. IFN-a1 was administered to the cells at a concentration of 10 U/mol in EMEM containing 2% FBS. Two hundred Ngami of recombinant rainbow trout IFNγ  were used for stimulation of the cells.
Cloning of STAT1 and plasmid constructs
Specific primers for amplifying the salmon STAT1 gene were made based on the rainbow trout (Oncorhynchus mykiss) STAT1 sequence with the GenBank accession number U60331. The primers are listed in Table 1. The primers were Gateway compatible allowing the PCR fragment to be inserted into pENTR/D-TOPO vector (Invitrogen). A 2.3 kB fragment was amplified by Pfu DNA polymerase (Stratagene) using mixed cDNA from salmon ovary and HK obtained as described earlier . Constructs were verified by DNA sequencing using the BigDye chemistry and a 3100 Gene Analyzer (Applied Biosciences). For transfection in cells, inserts were further transferred to the Gateway compatible eukaryotic expression vectors pDEST12.2 (Invitrogen), pDEST-GFP or pDEST-Myc (both provided by Dr. T. Lamark, University of Tromsø) by Gateway recombination using LR clonase II enzyme mix (Invitrogen) following manufacturer's instructions. For yeast two-hybrid analysis modified Clontech vectors (pGADT7 and pGBKT7) were used. The vectors (kindly provided by Dr. O. M. Seternes, University of Tromsø) were made Gateway compatible by insertion of the Gateway polylinker region as described  and are named pDESTGal4AD and pDESTGal4DBD respectively. Recombination into pGal4DBD required an intermediate cloning step into the pDONR207 vector (Invitrogen). Control plasmids pTD1-1, pGBKT7-53 and pGBKT7-Lam were purchased from Clontech.
Alignment of different STAT1 protein sequences from Atlantic salmon (ssSTAT1a, EU016199 and BT045567), Rainbow trout (U60331), Snakehead (EF079868), Green pufferfish (AF307105), Japanese flounder (EF491182), human (NM_007315), rat (NM_032612), African clawed frog (Xenopus, AY101602), Zebrafish (NM131480), Crucian carp (AY242386), mouse (NP_033309), pig (NP_998934), cow (NP_001071368), and chicken (NP_001012932). In addition other STATs (STAT2, STAT3, STAT4, STAT5 and STAT6 from some of these species were included in the analysis done by BioEdit and ClustalW version 1.81. A phylogenetic tree of STAT1 proteins was constructed using the neighbor-joining algorithm in clustalW. Bootstrap values were set to 1000.
Real-time RT-PCR quantification
Total RNA was extracted from head kidney leukocytes using RNeasy® Mini Kit (Qiagen) or from salmon organs using TRIzol® (Invitrogen). RNA (300 ng in a 20 μl reaction) was reverse transcribed using TaqMan® Reverse Transcription Reagents (Applied Biosystems). A volume of 2 μl of cDNA (6.25 ng of reverse-transcribed RNA) per 25 μl PCR reaction was used with primers for ssSTAT1 (ssSTAT1fw and ssSTAT1rev, see table 1) together with an ssSTAT1 probe (5'-6FAM-ACCACCAAGGAATGTTC-3'). While 6.25 pg of reverse-transcribed RNA per 25 μl PCR reaction was used to estimate 18S rRNA levels. The expression of mRNA was measured in an ABI Prism 7500 FAST Cycler (Applied Biosystems) using custom TaqMan® assays designed by Applied Biosystems and FAST PCR mastermix (Applied Biosystems). The amplification profile was 95°C for 20 s followed by 40 cycles of 95°C for 3 s and 60°C for 30 s. All cDNA samples were performed in triplicates. The expression was normalized against EF1AB and presented as relative expression compared to the non-treated control sample. Relative expression was calculated using the Pfaffl's mathematical model . The expression profiles from non-treated control fish organ samples were presented as expression of STAT1/EF1AB.
Both rich and selective yeast growth media were made from commercially available powders (Clontech). Yeast cells were grown at 30°C for 2 - 4 days. Plasmid constructs based on the pGal4DBD vector or the pGal4AD vector were transformed using Frozen-EZ Yeast Transformation II kit (Zymo Research) into competent yeast cells of strains S. cerevisiae Y187 (MATα) or PJ69-2A (MATa) by selecting for growth on medium lacking leucin or tryptophan, respectively. At least ten of each transformants carrying STAT1 were mated to each other. Ten diploid yeast cells from the mating were plated and scored for growth on a triple drop out medium (TDO) lacking leucin, tryptophan and histidine or a quadruple drop out medium (QDO) lacking leucin, tryptophan, histidine and adenine. Growth on TDO plates indicates a weak interaction, whereas growth on QDO plates indicates a stronger interaction. SV40 T-antigen (pTD1-1) and p53 (pGBKT7-53) served as positive controls, LaminC (pGBKT7-Lam) and empty vectors as negative controls.
For transfection, HEK-293, CHSE-214 or TO cells were seeded into 24-well plates with a density of 2 × 105 cells/well for HEK293 and CHSE-214, and 1 × 105 cells/well for TO, while in 6-well plates the densities were 1 × 106 or 5 × 105 cells/well, respectively. The cells were transfected the next day at 80 - 90% confluence. Transfection of the HEK-293 and CHSE-214 cells was performed by using the Lipofectamine 2000 (Invitrogen) transfection reagent according to the manufacturer's protocol. For each well, a total of 0.8 μg of plasmid DNA was incubated with 2 μl Lipofectamine 2000 in 100 μl serum-free EMEM for 20 min at room temperature before added to the cells. Three hours post transfection, FBS was added to a total concentration of 2%. For transfection of the TO cells the transfection reagent FuGENE HD (Roche Applied Science) was used according to the manufacturer's protocol. A total of 0.6 μg of plasmid DNA was mixed with 1.25 μl FuGENE HD in 50 μl EMEM and incubated 15 min before added to the cells with medium containing 2% FBS.
TO cells transiently transfected with a Mx-promoter-luciferase construct  were lysed in 50 μl 1× passive lysis buffer (PLB), from the Dual-Luciferase Reporter Assay System (Promega). 100 μl of luciferase assay buffer II (LAR II) was predispensed in a luminometer plate (96 wells) and 20 μl of the lysate added (according to the manufacturer's protocol). The firefly luciferase activity  was measured in a Luminoscan RT luminometer (Labsystems OY), before 100 μl of Stop&Glo reagent was added and Renilla luciferase activity recorded for estimation of transfection efficiency (LAF/LAR). All samples for the luciferase assay were set up in triplicate and the results given as relative light units (RLU).
NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo scientific) was used to extract nuclear and cytoplasmic proteins from 2 × 106 TO cells pr. reaction according to the manufacturer's protocol.
Gel electrophoresis, Western blotting and antibodies
Cells were lysed in 50 μl sodium dodecyl sulfate (SDS) sample buffer (160 mM Tris-HCl [pH 6.8], 10% β-mercaptoethanol, 2% SDS, 20% glycerol, 0.1% bromophenol blue), transferred from the culture well into a microcentrifuge tube and boiled for 5 min. Then, typically 15-20 μl of the samples were loaded in each well of a precast 4-12% gradient NuPAGE Novex Bis-Tris gel and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with MES buffer and Western blotting using the Invitrogen NuPAGE system. Gel electrophoresis, blotting, blocking and antibody incubation were performed as described by the manufacturer. A polyclonal antibody against STAT1 (α-STAT1) was custom made from a C-terminal peptide retrieved from the salmon STAT1 sequence: RSVAPVFQCWTGPKE. The peptide was cross-linked by BSA to glutaraldehyde and two rabbits were injected with antigen every 14 days. The terminal bleed of the rabbits took place after day 68 (5 injections). The antiserum was antigen-purified and reactivity against the peptide was confirmed by dot blot analysis against the peptide. The specificity of the antiserum was checked by transfection and expression of a STAT1-GFP fusion construct in different cell-types and the results are presented as supplementary data (Additional file 3, supplemental Figure S2). A dilution of 1:2000 of the anti-STAT1 antibody (α-STAT1) was found to be appropriate for Western blotting. A polyclonal Mx antibody (α-Mx, 1:1000 dilution)  was applied as primary antibody for detection of Mx protein, and a GFP antibody (1:10000 dilution) (Abcam) for detection of GFP-tagged proteins. An actin antibody (1:1000 dilution) produced in rabbit (Sigma) was used as loading control in most Western blots and an antibody directed towards cod Cathepsin D (1:2000 dilution)  was used as a cytoplasmic marker. Goat anti-rabbit-Horseradish Peroxidase (HRP) antibody or goat anti-mouse-HRP antibody (Santa Cruz Biotechnology) diluted 1:25000 were used as secondary antibodies. Detection was performed by using SuperSignal West Pico chemiluminescent substrate (Pierce Biotechnology Inc.). Stripping of the membranes was performed in 0.2 M NaOH for 10 min followed by washing, blocking and new antibody incubation.
Immunoprecipitation (IP) of STAT1
TO cells or Atlantic salmon spleen and HK leukocytes were stimulated with IFN-a1 or IFNγ for 1 and 3 h before washed two times with ice-cold PBS and harvested in buffer A (20 mM Tris-acetate, pH 7.0; 0.27 M sucrose; 1 mM EDTA; 1 mM EGTA; 1 mM orthovanadate; 10 mM β-glycerophosphate; 50 mM sodium fluoride; 5 mM sodium pyrophosphate; 1% [vol/vol] Triton X-100; 0.1% [vol/vol] 2-mercaptoethanol) and 'Complete' protease inhibitor cocktail (one tablet/50 ml, Roche). Lysates were cleared by centrifugation at 4°C for 15 min at 18000 × g. Lysates were then subjected to IP by incubating for 1 h at 4°C with αSTAT1 (1:100), before addition of 10 μl protein A-agarose (50% slurry pre-equilibrated in buffer A) and incubation at 4°C for 1 h. The immunoprecipitated material was washed four times in ice-cold buffer A with 0.5 M NaCl and resuspended in 40 μl 2× LDS-sample buffer. STAT1 was detected by the tyrosine phospho-specific antibody 4G10 Anti-Phosphotyrosine (Millipore) after SDS-PAGE and Western blotting.
HEK-293 cells co-transfected with the eukaryotic expression vectors pEXP12.2-STAT1 and pEXP-GFP-STAT1 or pEXP12.2-STAT1 and pEXP-GFP were washed two times with ice-cold phosphate-buffered saline (PBS) and harvested in HA-lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 2 mM EDTA, 1 mM EGTA, 1% Triton X-100 ) with a protease inhibitor cocktail added (Complete EDTA-free, Roche). Cell lysates were incubated on ice for 15 min and cleared by centrifugation for 15 min at 18000 × g in a microcentrifuge. Lysates were then subjected to IP with either α-STAT1 or α-GFP together with pre-blocked Protein A/G PLUS-Agarose beads (Santa Cruz biotechnology). The agarose beads were then washed four times with HA-lysis buffer, and all traces of buffer removed with a pipette tip before elution in 50 μl 2× SDS sample buffer. Eluted proteins were subjected to SDS-PAGE and visualized by Western blotting and antibody detection.
To examine localization of STAT1 in cells, primary cells from salmon HK were seeded on 14 mm coverslips. Non-adherent cells were washed away 1 day after seeding, and adherent monocyte/macrophages were stimulated with recombinant IFNs as described above. The cells were rinsed with 1× PBS (phosphate-buffered saline), before fixed using 4% paraformaldehyde for 20 min at room temperature. Cells were washed three times with PBS, and permeabilized with 0.3% Triton X-100 for 15 min, before blocked for 30 min with 7.5% FBS in PBS. The cells were then incubated with the primary antibody (α-STAT1) at a 1:500 dilution in PBS 7.5% FBS for 1 h washed three times with PBS and incubated with secondary antibodies conjugated to Alexa Fluor 546 at a 1:2000 dilution (Invitrogen) for 45 min away from light. Cells were washed and stained with DAPI (4',6'-diamidino-2-phenylindole, 1:300, Invitrogen) and mounted on slides with an antifade mounting medium. Confocal laser scanning microscopy was performed using a Leica TCS SP5 confocal microscope with LAS AF software.
This work was financed by the National Program for Research in functional Genomics in Norway (FUGE) of the Research Council of Norway (grant no 159326/S10). Dr. Cheng-Yin Shi was supported by a cultural agreement between Norway and China scholarship. We would like to thank Professor B. Robertsen, University of Tromsø, who kindly provided the Mx-antibody and the recombinant IFN-a1. The rIFNγ was a generous gift from Professor C. J. Secombes, University of Aberdeen, UK, and the TO cells from Professor H. Wergeland, University of Bergen. The CatD antibody was provided by P.A. Wang, University of Tromsø. The technical assistance from Dr. D.B. Iliev in providing primary leukocytes is appreciated. Dr. I. Skjæveland contributed to the phylogenetic analyses of the STATs.
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