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Cell Communication and Signaling
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Potential cellular conformations of the CCN3(NOV) protein Stanimir Kyurkchiev, Herman Yeger, Anne - Marie Bleau and Bernard Perbal*
Address: Laboratoire d’ Oncologie Virale et Moléculaire, UFR de Biochimie, Université Paris 7-D. Diderot, Paris, France
Email: Stanimir Kyurkchiev - kyurkch@hotmail.com; Herman Yeger - hermie@sickkids.on.ca; Anne - Marie Bleau - bleau@ccr.jussieu.fr; Bernard Perbal* - perbal@ccr.jussieu.fr
* Corresponding author
| Published: 10 September 2004 | Received: 26 August 2004 |
|---|---|
| Cell Communication and Signaling 2004, 2:9 doi:10.1186/1478-811X-2-9 | Accepted: 10 September 2004 |
This article is available from: http://www.biosignaling.com/content/2/1/9
@ 2004 Kyurkchiev et al; licensee BioMed Central Ltd.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Aim: To study the cellular distribution of CCN3(NOV) and to determine if the carboxyterminus of CCN3 is hidden or masked due to high affinity interactions with other partners. CCN3 was detected using affinity purified antibodies (anti-K19M-AF) as well as a Protein A purified anti-K19M antibodies (anti-K19M IgG) against a C-terminal 19-aminoacid peptide (K19M) of human CCN3 protein. The antibodies were applied in indirect immunofluorescence tests and immunoenzyme assays on glial tumor cell line, G59, and its CCN3-transfected variant G59/540 and the adrenocortical cell line, NCI-H295R.
Results: Anti-K19M-AF antibodies reacted against K19M peptide in ELISA and recognized two bands of 51 kDa and 30 kDa in H295R (adrenocortical carcinoma) cell culture supernatants by immunoblotting. H295R culture supernatants which contained CCN3 as shown by immunoblotting did not react with anti-CCN3 antibodies in liquid phase. Anti-CCN3 antibodies stained the surface membranes of non-permeabilized H295R and cytoplasm in permeabilized H295R cells. Similarly, anti-CCN3 stained surface membranes of G59/540, but did not react with G59 cells. Prominent cytoplasmic staining was observed in G59/540, as well as the cell footprints of G59/540 and H295R were strongly labeled.
Conclusions: The K19M-AF antibody directed against the C-terminal 19-aminoacid peptide of CCN3 recognized the secreted protein under denaturing conditions. However, the C-terminal motif of secreted CCN3 was not accessible to K19M-AF in liquid phase. These anti-CCN3 antibodies stained CCN3 protein which was localized to cytoplasmic stores, cell membranes and extracellular matrix. This would suggest that cytoplasmic and cell membrane bound CCN3 has an exposed C-terminus while secreted CCN3 has a sequestered C-terminus which could be due to interaction with other proteins or itself (dimerization). Thus the K19M-AF antibodies revealed at least two conformational states of the native CCN3 protein.
Introduction
The CCN3 protein belongs to an emerging family of growth regulators referred under the CCN acronym
(cysteine-rich protein, Cyr61, connective tissue growth factor, CTGF, and the nephroblastoma overexpressed gene, nov; CCN 1-3 respectively) [1-3]. The CCN family
now comprises six identified members with properties of both positive and negative regulators of cell growth, shar- ing a common multimodular organization. New mem- bers of the CNN family have been described over the past few years, and recent reviews on the CCN proteins high- light their intimate involvement in a variety of key biolog- ical processes including development, angiogenesis, and cancer [1-4].
The CCN3 (NOV) gene had been initially characterized as an integration site for the myeloblastosis associated virus MAV [5] which induces kidney tumors resembling neph- roblastoma and Wilms tumor [6]. In human and animal tumors, the expression of the CCN3 gene was found to be altered either positively or negatively [7-11]. Experiments performed in our laboratory have established that CCN3 is a marker of tumor differentiation in Wilms tumors [12] and several other tumor types [unpublished observa- tions]. Furthermore, an increasing amount of results assigns growth inhibitory functions to CCN3 in several conditions ([7,8,13-15], Manara et al. submitted).
The CCN proteins share a strikingly conserved multimod- ular organization with distinctive functional features [1]. From the amino to the carboxy terminus of these proteins, four modules can be recognized : an insulin-like growth factor (IGF) binding protein (IGFBP)-type motif, fol- lowed by a Von Willebrand type C (VWC) domain likely responsible for oligomerisation, a thrombospondin type 1 (TSP1) repeat, responsible for interaction with extracel- lular matrix proteins, and a carboxy-terminal module (CT), postulated to represent a dimerization domain, as it contains a cysteine-knot motif that is present and involved in the dimerization of several growth factors such as nerve growth factor (NGF), transforming growth factor -2 (TGF-2) and platelet derived growth factor BB (PDGFB).
The multimodular structure of CCN3 and other CNN pro- teins raises interesting questions as to participation of each individual module in conferring the biological prop- erties to the full length proteins. Either the biochemical functions of the individual IGFBP, VWC, TSP and CT modules are indeed conserved and in sum determine the ultimate function of the full length protein, or each mod- ule confers on the whole protein specific biological func- tions which may vary from the conserved function, and either substitute or add to those of individual modules.
Application of the yeast two-hybrid system and co-precip- itation strategies to identify proteins interacting with CCN3 has revealed that full length CCN3 interacts with several receptors, signaling molecules, and proteins of the extracellular matrix (16-19), suggesting functional
involvement of CCN3 in cell signaling and adhesion regulation.
Our results also established that truncated isoforms of CCN3 could bind specific targets and pointed the CT domain of CCN3 as a critical determinant for protein interaction. This led us to hypothesize that truncated iso- forms of CCN3 could also modulate its biological activity (3). The question therefore arises whether different con- formational states exist due to multiple protein interac- tions and thereby the presentation of known antigenic epitopes.
In the present study we have used an immunological approach to establish the cellular distribution of CCN3 in cell lines representing adrenocortical and glioblastoma tumors and to ask whether the CT module of CCN3 exists in different conformational states depending on its involvement in protein interactions and cellular location. We now provide evidence that the CT end of CCN3 exists in more than one conformational state.
Results
Cell culture supernatants and cellular lysates from the H295R, G59/540, and parental G59 cell lines were elec- trophoresed under denaturing conditions and immunob- lotted with anti-K19M IgG antibody. Immunoblot analysis revealed secreted forms of CCN3 for the H295R and G59/540 cell lines, consisting of two distinct bands at 51 kDa and 30 kDa [Figure 1A]. The latter likely corre- sponded to the previously described amino-truncated CCN3 isoform [3]. Intracellular CCN3 proteins were also detected in these cell lines. However, in addition to the two bands at 48 kDa and 30 kDa, two other high molecu- lar species reacting positively with the antibodies were also detected in the lysates [Figure 1B]. The different sizes of these various isoforms likely results from post-transla- tional modifications and oligomerisation of CCN3 protein.
When tested in ELISA, pre-incubation of the anti-K19M- AF antibodies with CCN3-containing H259R supernatant did not affect the binding of anti-K19M-AF antibodies to the K19M peptide coated on microtitre plates (Figure 2). Under identical conditions, the absorption of K19M-AF antibodies with serial dilutions of K19M peptide showed a dose-dependent absorption pattern with 7.78 ug/ml K19M peptide, yielding a 50% reduction in the binding of anti-K19M-AF to K19M peptide coated on plates (Figure 3). These results suggested that the K19M-AF antibodies did not recognize the CCN3 protein in its native configu- ration, whereas it can be detected in the same sample after denaturation and Western blotting.
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When fixed and non-permebialized cells were used in cell- ELISA with anti-K19M-AF antibodies it was shown that positive reaction of the antibodies could be recorded with H295R cells which are known to synthesize and secrete CCN3 protein, while the reaction with G59 cells was in the ranges of the negative background. After permebialization of the cells the intensity of the reaction was increased but a significantly positive reaction was recorded with H295R cells (Figure 4).
Since CCN3 was secreted by H259R cells, it was important to check whether it could bind cell surface. Evidence for this would lend support to the previous suggestion of an autocrine mechanism of control by the CCN3 protein. To explore this possibility cells from H259R, G59/540 and G59 cell lines were incubated for 1 h on ice in the presence of supernatant containing CCN3 protein and then ana- lyzed by cell ELISA as described above. The results obtained showed that such a treatment did not increase the intensity of the reaction with anti-K19M-AF bound to the cell surface. These experiments demonstrated that
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H295R, and G59/540 which expressed CCN3 on the cell surface and no further absorption occurred, and the control G59/540 cells did not absorb CCN3 from the cul- ture supernatant (data not shown).
Cellular Localization of CCN3
Paraformaldehyde fixed, non-permeabilized H259R cells treated with the anti-K19M antibody (Protein A purified) exhibited immunofluorescent membrane specific staining distributed over the cell surface (Figure. 5A) while simi- larly treated G59 CCN3 negative cells did not stain (not shown). The CCN3-transfected glioblastoma cell line G59/540 stained positively with a similar localization of the reaction product (Figure 5B - G540). Interestingly, since cells grown on coverslips and fixed in paraformadehyde tend to slough, we did note the pres- ence of positively staining cell footprints, suggesting dep- osition of CCN3 protein in a secretable extracellular matrix (Figures 5C,5D). After ethanol/formalin fixation and further permeabilization of the cells with 0.1% Triton X-100 the anti-K19M antibody (Protein A purified) gave an intensive granular fluorescence pattern which appeared perinuclear in a significant fraction of the cells with a similar pattern observed in H295R and G59/540 cells (Figure 5E,5F). On the other hand, the parent G59 cell line showed a weak, but still perinuclear cytoplasmic staining (Figure 5G). The latter may represent a smaller endogenous isoform of CCN3.
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In summary, the results produced using an immunologi- cal approach would suggest multiple conformations of the C-terminal end which harbors the immunogenic epitopes. Furthermore, these variations are associated with cell bound and secreted forms of CCN3. Cytoplasmic localization indicated abundant CCN3 protein localized with ER and Golgi networks.
Discussion
In this study we exploited the range of binding affinities present in polyclonal antibodies raised against the C-ter- minal peptide of CCN3 and analyzed with different immunoaffinity methods to ask whether CCN3 exists in alternate conformational forms in cell cultures and super- natants. Whereas immunocytochemistry of fixed, perme- abilized and non-permeabilized cells yielded evidence of both cell surface membrane and cytoplasmic expression and topographical distribution of native CCN3, ELISA method and western immunoblot revealed different pos- sible conformational forms of CCN3. Taken together the results of the assays suggest that native CCN3 assumes dif- ferent configurations that either expose or sequester the C- terminal peptide depending on whether CCN3 is cell associated or free within the culture supernatant. When considered in the light of recent evidence indicating that CCN3 can associate with specific integrins at the cell membrane [23,24] the question also arises whether CCN3 associates with specific protein partners in the circulation
and in the extracellular matrix produced by different cell types.
In these studies we focused on two cell lines, H295R and G59, representing adrenocortical and glial tissues, signifi- cantly different in their anatomical location and microenvironment. Since CCN3 has been demonstrated in plasma [25] it is conceptually feasible that CCN3 may be secreted by well organized ectodermal, mesodermal and endodermal cell types where it is expressed [26-28], and then is transported through complex extracellular matrix to enter the circulation. Moreover, as CCN3 has been shown to be expressed by endothelium [23,29], the source of the circulating CCN3 may be restricted to endothelium. Keeping to this scenario, tissue expression of CCN3 would be restricted to regional cell types and its arena of activity relegated to the extracellular matrix and resident cells. Interestingly, we could show in vitro that CCN3 is sequestered in cell footprints representing a secreted extracellular matrix. Cell footprints can be depos- ited by various cell types in different arrangements and consists of extracellular matrix, including a variety of base- ment membrane proteins [30-32]. In our studies we noted a more uniform and punctuate deposition of CCN3 in the footprints suggesting possible association with regularly arranged clustered partners (e.g. integrins), yet to be deter- mined. Localization of CCN3 to footprints is perhaps expected since it has been shown to mediate adhesion of endothelial cells [23], in turn triggering intracellular phos- phorylation signaling events.
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This then raises the notion that proximity of CCN3 to cell surfaces could allow CCN3 to function in possible auto- crine and paracrine mechanisms. Depending on the asso- ciating proteins, CCN3 would likely undergo specific conformational changes with potentially different func- tional outcomes. A variety of functional states may exist since CCN3 is expressed in secretable and non-secretable
isoforms and contains motifs that overlap with other pro- teins and therein, additional binding partners [1,4]. Thus far the actual molecular function of native CCN3 has not been determined, although biologically it shows evidence of being able to regulate mitogenesis and motogenesis [3]. In turn, CCN3, compared to other CCN proteins, may be differentially regulated by mechanical stress [29]. As a
heparan binding protein, CCN3 could associate with a large group of molecules at the cell membrane and in the extracellular matrix. Yeast two hybrid studies have indicated associations with fibulin 1C [16]. Other studies have shown CCN3 involved in calcium signaling [19,33], associated with Notch signaling [17], and able to trigger membrane mediated phosphorylation events [34] Some of the cellular effects of CCN3 may also be mediated by different isoforms operating at the level of the nucleus [35].
The biological significance of different conformations of CCN3 is not known. However, examples from other studies have suggested that conformational changes can occur in serum proteins due to binding of bivalent cations [36]. Since we recently reported that CCN3 may interact with Ca2+ binding proteins like fibulin-1C, modulate calcium uptake, and considering that Ca2+ binding mod- ulates function of other protein partners such as integrins [reviewed in [33]], it is conceivable that secreted CCN3 could assume an altered conformation by binding biva- lent cations, directly or indirectly, like Ca2+ present in cul- ture media. Whether calcium or some other bivalent cation could be involved and how this could occur is still speculative as the sequence of CCN3 does not suggest any obvious cation binding properties. It is also possible that secreted CCN3 complexes with an as yet unknown partner thus sequestering the antigenic epitopes.
Altered expression of CCN3 in a variety of cancers may reflect maintenance of a normal homeostatic function of the cell of origin, or may indicate requirement of specific CCN proteins for maintaining the undifferentiated tumor state. One such example where the two possibilities are not yet resolved is in Wilms tumor, where CCN3 is abun- dantly expressed during normal nephrogenesis and in tumors [12]. Interestingly, CCN3 was originally identified during MAV virus induction of nephroblastoma but is not a direct target of WT1, the Wilms tumor suppressor gene [34]. Thus far few mutations have been described for CCN family and none for CCN3. CCN proteins have however been associated with a variety of cancers where they can be markedly overexpressed [11,37-39]. It may be that CCN proteins are not directly involved in tumorigenesis (e.g., Wilms tumor) but rather play supporting roles or may act as a negative regulator on malignant behavior reflecting their roles as integrators of cell-cell and cell-matrix com- munications. Thus having antibodies that can recognize the different isoforms of CCN proteins with great specifi- city and in respect to specific epitopes within the domains would be invaluable for quantitation [40] and for dissect- ing their functions in communication signaling.
Conclusions
Our preliminary investigations here have revealed possi- ble physical and functional states of native CCN3 localizing to cytoplasmic, cell membrane and extracellu- lar matrix. Further complexity is added since shorter and larger isoforms of CCN3 can be detected using western blotting. The origin of these short forms is still not fully understood. As there do not appear to be alternate tran- scripts [1] this suggests post-translational processing including, in addition to variant glycosylation, phosphor- ylation, specific proteolytic events and sites. The C-termi- nal antibody recognizes these forms. The use of antibodies to other motifs in CCN3 will permit us to track the cleaved N-terminal peptide which potentially could be functionally active as it resembles IGFBP [1]. Therefore the cleavage products of CCN3 in concert with native CCN3 may also be involved in several aspects of the regu- lation of growth factor activity at the cell membrane or its management in extracellular repositories.
Finally, cells can coordinately express a variety of CCN proteins that are closely related, for example CCN1-3 with cross-over and opposite functional effects yet bearing similar functional domains. Evidence is starting to surface that they might compete for binding partners, such as integrins, thus forming protein complexes with different biological consequences to cell behavior [18,19]. It will be important to understand how stoichiometric changes in CCN protein concentrations can change the behavior of cells, thus opening up opportunities for therapeutic manipulations in disease. It is obvious that there will be a necessity for antibody reagents and quantitative method- ologies to enable these studies.
Materials and methods Cell Lines
NCI H259R (American Type Cell Collection) is a human adenocortical carcinoma cell line and was cultured in DMEM/F12 supplemented with 2.5 % Nu-serum plus ITS+ supplement (Sigma Co, St. Louis, USA). H295R cells have been characterized and were shown to secrete high levels of CCN3 protein [20,21]. The glioblastoma cell line (G-59) has been described previously [22]. CCN3 express- ing G59/540 sublines were obtained following transfec- tion of G59 with pCMV CCN3 plasmid and G418 selection [13]. These cell lines and their derivatives were used in cell ELISA and indirect immunofluorescence labe- ling experiments as described below.
Antibodies
Antibodies against C-terminal peptide K19M were used in these experiments after either purification by an antigen specific affinity chromatography (anti-K19M-AF) or by Protein A chromatography (anti-K19M IgGs).
Antibody Affinity Purification and Characterization
The K19M C-terminal peptide (KNNEAFLQELELKT- TRGKM) of human CCN3 protein was coupled to CNBr activated Sepharose 4B (Pharmacia Biotech, Uppsala, Sweden) following the protocol recommended by the manufacturer. Briefly, 3 mg of peptide were dissolved in 5 ml of 0.1 M NaCO3 pH 9.0 containing 0.5 M NaCl (cou- pling buffer) and added to 3.5 ml CNBr-Sepharose swelled gel and the mixture was rotated end-over-end for 2 hours at room temperature. Excess ligand was eluted with 20 ml of coupling buffer and the gel was incubated in 0.1 M Tris-HCl buffer pH 8.0 for 2 hours at room tem- perature. The gel was washed 5 times in cycles consisting of 20 ml of 0.1 M acetate buffer pH 4.0 followed by 20 ml of 0.1 M Tris-HCl buffer pH 8.0, each containing 0.5 M NaCl, and then packed into a PD-10 column.
The rabbit anti-K19M antiserum was absorbed with 1 mg/ ml human serum albumin to remove cross-reactivity with human plasma and dialyzed overnight at 4℃ against phosphate buffer pH 7.0. An aliquot of 3.5 ml serum was loaded on the affinity column and the flow through and the unbound proteins were collected in 3 ml fractions fol- lowed by thorough washing of the column with the load- ing buffer. The K19M peptide bound antibodies were eluted with 9 ml of 0.1 M Tris-glycine buffer pH 2.8 in 3 ml fractions that were collected into test tubes containing 100 ul 1 M Tris buffer pH 8.0. All column fractions were tested by ELISA for the presence of antibodies reacting against K19M peptide and the positively reacting fractions were further purified by affinity chromatography on pre- packed HiTrap Protein A columns (Pharmacia Biotech, Uppsala, Sweden) as recommended. Affinity purified antibody preparations were further tested to determine their reactivities and specificities. The affinity purified anti-K19M-AF antibodies reacted against the K19M pep- tide when tested in serial dilutions in ELISA (see below). The titers of K19M-AF antibodies were comparatively lower as compared to the unfractionated K19M antise- rum. This finding was not unexpected as it likely reflects the polyclonal composition of the primary rabbit antise- rum and differences in the content of the specific mono- clonal specificities in the antiserum. Importantly, the affinity purified antibodies recognized the K19M peptide when coated on a solid phase.
K19M Peptide Enzyme-linked Immunoabsorbent Assay (ELISA)
Affinity purified antibodies were titered by ELISA. Individ- ual wells of polystyrene 96-well flat bottom plates (NUNC) were coated with 1 ug/ml of K19M peptide diluted in coating buffer (0.05 M carbonate buffer pH 9.6) by incubation overnight at 4℃. The unsaturated protein binding sites were blocked with 300 ul/well 2% BSA for 1 h at room temperature. The primary anti-K19M antiserum
and affinity purified antibodies were added in serial dilu- tions in duplicates and the wells were incubated for 2 h at room temperature. After thorough washing the wells were incubated with goat anti-rabbit IgG serum conjugated with peroxidase (Sigma Co) diluted 1/5000 in blocking buffer for 1 h at room temperature. The bound enzyme activity was revealed by adding the enzyme substrate 0.5 mg/ml ortho-phenylenediamine in citrate buffer pH 5.0 containing 0.5 ul/ml H202. The enzyme reaction was stopped by addition of 50 ul/well of H2SO4 and the color reaction was read at 492 nm in a MicroELISA reader.
Cell Enzyme-Linked Immunoabsorbent Assay (Cell ELISA)
Tumor cell lines were cultured in complete medium in 96- well flat bottom plates (Corning) to form a subconfluent monolayer and further incubated overnight in serum free medium. The cells were washed 3 times for 5 min each with phosphate buffered saline (PBS, pH 7.2) and cells were fixed by treatment with ice-cold methanol for 30 min warming to room temperature. The endogenous per- oxidase activity was blocked with 3% H2O2 in distilled water for 7 min at room temperature followed by 3 x 5 min washes in PBS, pH 7.2. Non-specific binding was blocked with 1% bovine serum albumin (BSA) for 1 h at room temperature. Cells were washed once in PBS and incubated with K19M-AF diluted in 1%BSA for 2 h at room temperature. After 3 × 10 min washes in PBS goat- anti rabbit IgG conjugated with peroxidase diluted 1/ 10000 in 1% BSA-PBS was added for 1 hour at room tem- perature. The cells were washed again in PBS and the bound enzyme activity was developed by adding ortho- phenylendiamine (5 mg/10 ml citrate buffer, pH 5.0) containing 5 ul of 30% H2O2. The color reaction was stopped by adding 50 ul of 10% H2SO4 and the intensity was read at 492 nm in a MicroELISA reader.
Gel Electrophoresis and Western blotting
To prepare proteins for immunoblotting, cells were lysed in NP40 buffer (50 mM Tris hydrochloride, pH 8.0, 150 mM NaCl, 5 mM EDTA and 2% NP40) with protease inhibitors (Cocktail Tablets, complete, Roche) and phos- phatase inhibitors (50 mM NaF, 2 mM sodium orthovanadate) for 30 min at 4℃. After centrifugation at 15 000 g, extracts were stored at -80℃ until use. CCN3 proteins in the conditioned medium were concentrated on Heparin Sepharose (Amersham, Uppsala, Sweden) as described by Chevalier et al (1998). Briefly, supernatants were incubated overnight with heparine and then washed 4 times in PBS containing protease inhibitors. Bound CCN3 was dissociated using 2-mercaptoethanol in Lae- mmli buffer, boiled for 10 min and then centrifuged to keep the free protein. Heparin Sepharose concentrated samples and cellular extracts were subjected to electro- phoresis under reducing conditions in 12.5% polyacryla- mide gels. Separated proteins were subsequently
transferred to nitrocellulose by a semi-dry blotter (LKB Biotech, Sweden) as recommended by the supplier. The nitrocellulose sheet was blocked by incubation for 1 hour at room temperature with 5% nonfat milk in PBST (PBS with 0.2% Tween 20, pH 7.4). The membrane was then incubated in the same buffer with the anti-K19M IgG (1/ 2000) and then washed extensively. The blots were further incubated in goat anti-rabbit IgG conjugated with peroxi- dase (1/10 000 in blocking solution, Sigma Co, USA) for 1 hour at room temperature. Revelation was performed using the chemoluminescence protocol and reagents (Pierce, Rockford, IL, USA).
Indirect Immunofluorescence Labeling
Cells were grown on alcohol flamed coverslips, rinsed in PBS and fixed in cold 70% ethanol containing 10% for- malin (Sigma) for 10 min on ice, and stored in PBS. Immunofluorescence labeling was performed at room temperature. For this procedure, coverslips were placed into weighing boats [Sigma; 4.5cm × 4.5cm] maintaining cell side up. Cells were further permeabilized in 0.5% Tri- ton X-100 in PBS for 15 min and then blocked in 5%FBS/ PBS for 30 min.
Anti-K19M IgGs antibodies were applied at 1:1000 dilu- tion in 5%FBS/PBS for 1 hour with intermittent rotation, followed by 5 washes in PBS containing 0.1% Tween 20. Subsequently, the cells were incubated in anti-rabbit IgG serum conjugated with either Alexa 488 (green fluores- cence) or Alexa 594 (red fluorescence), in 5%PBS/BSA for 1 hour. After final washes in PBS/Tween 20 followed by one wash in PBS cells were mounted with antifade mount- ing medium (Bio-Rad, France), excess liquid adsorbed with filter paper, and coverslips sealed with clear nail polish. Immunofluorescence images were captured on 400 ASA film and processed further with Adobe Pho- toshop (version 7.0).
List of abbreviations
None.
Competing interests None declared.
Authors’ contributions
SK carried the affinity chromatography and immunoen- zyme experiments;
HY carried out immunofluorescence labeling experiments;
AMB carried out IgG purification and Western blots;
BP conceived the study design and coordinated and edited the manuscript
Acknowledgements
This study was supported by funds to BP from Association pour la Recher- che Contre le Cancer (ARC), Ligue Nationale Contre le Cancer (Comités du Cher et de l’Indre), and Ministère de l’Education Nationale, de la Recher- che et de la Technologie. Part of this work has been achieved when the Lab- oratoire d’Oncologie Virale et Moléculaire was affiliated to the INSERM. SK and HY were invited Professors of the Université Paris 7-D. Diderot. Per- manent address for SK : Department of Molecular Immunology, Institute of Biology and Immunology of Reproduction, Bulgarian Academy of Sciences, Sofia, Bulgaria. Permanent address for HY : Department of Paediatric Lab- oratory Medicine, The Hospital for Sick Children and Laboratory Medicine & Pathobiology, University of Toronto, Toronto, Canada. AMB is recipient of a fellowship from the Fonds de la Recherche en Santé du Québec.
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