Glycobiology, 2000, Vol. 10, No. 4 413-419
© 2000 Oxford University Press
Regulation of CD45-induced signaling by galectin-1 in Burkitt lymphoma B cells
Université Paris 13, Biochimie Cellulaire des Hémopathies Lymphoïdes, 93017 Bobigny, France; 2Laboratoire d’Immunologie Cellulaire, Centre de Transfusion Sanguine des Armées, 92140 Clamart, France; 3Institute of Genetics, Biological Research Center, 6726 Szeged, Hungary; and 4INSERM U 25, Hôpital Necker, 75015 Paris, France
Received on September 16, 1999; accepted on October 23, 1999.
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Abstract |
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It has been well established that Galectin-1 (GAL1), a ß-galactoside-binding protein, regulates the viability of lymphoid cells. However, the signaling pathway governed by the binding of GAL1 to the cell membrane is not understood. As a first step towards the elucidation of GAL1-initiated signaling events leading to a reduced viability of Burkitt lymphoma B cells, we tried to characterize the initial events induced by the binding of GAL1 to its receptor. This characterization was performed in BL36 cells, a Burkitt lymphoma cell line sensitive to GAL1. The results were as follows: (1) when solubilized cell membrane lysates were affinity bound to immobilized GAL1 and eluted by competition, the tyrosine phosphatase glycoprotein CD45 was found in the eluate, highlighting the role of CD45 as a receptor of GAL1; (2) the phosphatase activity of cell membranes diminished after incubation with GAL1; (3) immunoprecipitation experiments demonstrated that the phosphotyrosine kinase Lyn was dysregulated in cells that have been cultured in medium containing 700 nM GAL1, and (4) that the ratio between two isoforms of Lyn was modified during the treatment with GAL1. The regulation of Lyn therefore seems to be a key event in the action of GAL1.
Key words: Burkitt lymphoma/CD45/cell death/galectin/Lyn
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Introduction |
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GAL1 is a member of the galectin family of lectins defined by a conserved carbohydrate recognition domain showing affinity for ß-galactosides (Kasai and Hirabayashi, 1996
; Gabius, 1997; Perillo et al., 1998). Within the cells, GAL1 can be detected in the cytoplasm and the nucleus (Kuchler et al., 1989; Vyakarbam et al., 1998). Although functions for GAL1 have been proposed in each of these subcellular locations, it is also secreted by a non conventional pathway and is found on the cell surface (Cooper and Barondes, 1990; Avellana-Adalid et al., 1994; Lutomski et al., 1997). However, whereas the externalization of GAL1 and its binding to cell surface receptors are fairly well documented, its autocrine or paracrine functions have long remained elusive. Several lines of evidence indicate that GAL1 plays a role in the modulation of cell viability, as a regulator of the cell cycle or as a pro-apoptotic factor (Goldstone and Lavin, 1991; Wells and Mallucci, 1991; Allione et al., 1995; Perillo et al., 1995; Fouillit et al., 1998; Walzel et al., 1999). In the glucocorticoid-sensitive human T cell line CEM C7, GAL1 was found to be overexpressed during the induction of apoptosis (Goldstone and Lavin, 1991); and exogenously added GAL1 was observed to regulate the growth and death of activated T cells, thymocytes, and lymphoblastoid T-cell lines (Allione et al., 1995; Perillo et al., 1995; Fouillit et al., 1998; Vespa et al., 1999; Walzel et al., 1999). In Jurkat T cells, ligation of CD45 by GAL1 ultimately led to cell death in a concentration-dependent manner (Fouillit et al., 1998; Walzel et al., 1999). However, the definitive establishment of the role for GAL1 in regulation of cell viability and growth requires further investigation. As reported here, we tested the hypothesis that GAL1 modulates the viability of Burkitt lymphoma (BL) cells, and we investigated the first steps of the signal resulting from the binding of GAL1 to its cell membrane receptor. We found that the membrane receptor for GAL1 on BL cells is CD45, the leukocyte common antigen. Furthermore, we found that the binding of GAL1 to CD45 inhibits its protein phosphatase activity, and therefore the dephosphorylation of Lyn kinase. These results provide strong evidence that GAL1-CD45 interaction, and therefore the modification of Lyn phosphorylation, are required for the effect of GAL1 on B cells.
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Results and discussion |
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This study was initiated to examine the regulation of cell viability associated with the binding of GAL1 to its cell membrane receptor, and to determine which signaling events result from this binding in BL cells. GAL1 that is detected in EBV-transformed lymphoblastoid cell lines is down regulated in BL cells (unpublished observations). However, BL cells can be induced to express and secrete GAL1 by drugs that influence the expression of differentiation markers, in significant contrast to untreated cells. In addition, during serial propagation, the EBV-carrying BL lines often drift spontaneously towards a more lymphoblastoid-like phenotype associated with up regulation of GAL1 (F.Poirier et al., unpublished observations). The mechanism of this phenotype-dependent regulation of GAL1 expression, as well as the biological consequences of this expression, are not known. In this work, we examined the effect of added GAL1 on three BL cell lines (Table I). A relatively low concentration of GAL1, i.e., 700 nM, was used, according to previous results on the Jurkat cell line (Fouillit et al., 1998
; and unpublished observations). In BL36 cells, a drop in the viable cell number of about 28% was observed over the 24 h period following the addition of GAL1, and went up to 40% after 48 h. The degree of cell death was clearly dose-dependent and cell line-dependent, and was more limited in DG75 and BL31 cells than in BL36.
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The cells were then stained with merocyanin-540, a dye that incorporates into the membrane following loss of phospholipid asymmetry in apoptotic cells (Fadok et al., 1992
; Lesage et al., 1997). The membranes of GAL1-treated BL 36 cells bound increased amounts of merocyanine 540 dye relative to those of untreated cells at the end of a 24 h incubation period, indicating that their membrane lipids were more loosely packed, consistent with a loss of membrane phospholipid asymmetry (mean percentage of merocyanin positive cells: 22.3 ± 7.6).
Flow cytometry investigation for the presence of GAL1 receptors on the surface of BL36 showed that the binding of GAL1 to the cell surface was galactoside specific, since the staining with GAL1-biot/streptavidin-FITC was abolished when cells were incubated in a buffer containing 25 mM thiodigalactoside (Figure 1). The binding was strongly dependent on the temperature, a characteristic of the lectin activity of GAL1 (Caron et al., 1987). These results indicated that BL cells express GAL1 receptors on their surface, and that carbohydrate–lectin interaction is the primary mechanism that binds GAL1 onto the cell surface.
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It has been suggested that the tyrosine phosphatase glycoprotein CD45 could be a functioning GAL1 receptor in erythroleukemic and T cells (Perillo et al., 1995
; Lutomski et al., 1997; Walzel et al., 1999). Membrane proteins were therefore separated by SDS–PAGE, transferred to Immobilon-P, and hybridized with GAL1-biot, and anti-CD45 mAb. As shown in Figure 2A, the proteins recognized by GAL1 migrated with a mobility similar to that of the proteins detected by specific mAb directed against CD45. For clearer identification of the GAL1 receptor expressed on BL cells and detected by flow cytometry, solubilized cell membrane lysate was affinity bound to agarose-GAL1, biotinylated, and eluted by lactose. The eluate was resolved by SDS–PAGE, and immunoblotted using anti-CD45 mAb. The results demonstrate that CD45 was adsorbed on the gel and specifically eluted by a competitive sugar, and therefore binds to GAL1 (Figure 2B). Blotted proteins were also detected with Strep-HRP, that detected all the proteins bound to GAL1. Two polypeptides with an electrophoretic mobility similar to that of immunodetected-CD45 were primarily detected with Strep-HRP. In order to identify precisely these two polypeptides a depletion experiment was done with immobilized anti-CD45 on the GAL1-bound fraction. Effectively, the two polypeptides were immunoprecipitated by the specific anti-CD45 Ab and thereafter dissociated by boiling the complex in SDS sample buffer (Figure 2B). Moreover, to verify that CD45 is actually a cell surface ligand, and not only a major GAL1-binding glycoprotein in lysates or blots, the effect of an excess of CD45 specific Ab on the cell binding of GAL1 was determined. Antibody to CD45 was able to dissociate GAL1 binding to BL36 cells more efficiently than the specific GAL1 inhibitor thiodigalactoside (Figure 3). CD45 therefore is nothing less than the major GAL1 receptor on the membrane of BL36 cells.
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CD45 is one of the heavily glycosylated proteins in leukocytes. It is expressed as multiple isoforms, which are generated through alternative splicing of three extracellular domain exons (Thomas, 1989
; Justement, 1997). Studies of both N-linked and O-linked sugar chains revealed that they contain large amounts of poly(N-acetyllactosamine) units (Sato et al., 1993; Furukawa et al., 1998), the carbohydrate structure specifically recognized by GAL1 (Caron et al., 1990). As GAL1 can be externalized from hematopoietic cells and then binds to cell surface receptors (Lutomski et al., 1997), it seems reasonable to propose that GAL1 is a natural ligand directly responsible for CD45 ligation, and that its interaction with CD45 may be associated with the regulation of signal transduction. As a first step towards the elucidation of this regulation, membrane fractions from BL36 cells treated or not with 2 µM GAL1 were assayed for PTPase activity. The PTPase activity was decreased in treated cells, as shown in Figure 4.
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Previous studies showed that CD45 activates the protein tyrosine kinase activity in Src protein tyrosine kinases by dephosphorylation of a conserved tyrosine near their C-terminus (Tamir and Cambier, 1998
). This C-terminal regulatory tyrosine of the Src kinase Lyn is hyperphosphorylated in CD45-deficient B cells, providing the direct evidence that this site is a target of CD45 in B cells (Yanagi et al., 1996). To assess the tyrosine phosphorylation of Lyn, cell lysates of untreated and GAL1-treated BL36 cells were immunoprecipitated with anti-Lyn Ab. The inhibitor sodium orthovanadate (1 mM) was added to the lysis buffer to inhibit in vitro phosphatase activity after extraction. The immunoprecipitates were electrophoresed, blotted to PDVF membrane, and analyzed with antiphosphotyrosine Ab. As shown in Figure 5A, the phosphotyrosine staining of Lyn doubled in 2 min, while the amount of Lyn protein immunoprecipitated with anti-Lyn did not increase. Lyn is expressed as two isoforms generated through alternative splicing. Upon the treatment with GAL1 the variation of phosphorylation was only observed for the higher molecular mass isoform. By 10 min, the tyrosine phosphorylation of Lyn returned to the basal level observed in the untreated sample. Then, we tested the possibility that Lyn kinase activity was altered after the binding of GAL1 to its cell receptor. Five min of GAL1 treatment abolished Lyn kinase activity, as demonstrated by the change of in vitro phosphorylation of the exogenous substrate enolase (Figure 5B). The kinase activity was restored after 30 min of incubation. Thus Lyn appeared to undergo a marked phosphorylation during the initial 5 min of treatment of BL cells with GAL1, accompanied by a decreased in vitro kinase activity.
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The regulation of Lyn kinase by phosphorylation is complex in that there are two identified tyrosine phosphorylation sites: C-terminal phosphorylation is inhibitory and autophosphorylation is stimulatory. Results obtained in CD45-deficient B cells indicated that phosphorylation status on C-terminal tyrosine of Lyn is dominant for determining its kinase activity (Yanagi et al., 1996
). Our results suggest that Lyn is phosphorylated at the activating autophosphorylation site in nontreated cells, but at both sites in GAL1-treated cells. A first attempt to study Lyn modifications thoroughly combined immunoprecipitation and bidimensional electrophoresis. Lyn was resolved in several spots indicating the presence of posttranslational modifications on the polypeptide in addition to the alternative splicing (Figure 6). After the treatment with GAL1, only two spots were modified, these two spots showing a same molecular weight of ~58 kDa and a difference in isoelectric point of 0.3 units: 5.3 and 5.6. The treatment led to a decrease of the volume of the less acidic spot, and to an increase of the more acidic. This shift of pI was compatible with the addition of a phosphoryl group on the polypeptide. Taken together, our results suggest that GAL1-induced ligation results in the phosphorylation of one tyrosine, presumably the negative regulatory site, on the 58 kDa form of Lyn.
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It was recently reported that the ligation of CD45 on human T or B cells by certain mAbs induces cell death (Klauss et al., 1996
; Lesage et al., 1997). However, until now the putative physiological inducer of CD45 ligation remains unknown. Moreover, the cascades of signaling induced by the ligation have not been fully elucidated. The hypothesis that GAL1 is a biological ligand of CD45 and that it regulates CD45 activity, and therefore cell viability, is consistent with the reports that: (1) GAL1 can be exported from the cytoplasm of hematopoietic cells depending on their state of differentiation or activation, and is able to bind to receptors on the cell surface by an autocrine mechanism (Lutomski et al., 1997); (2) the binding of GAL1 induces a rest in the cell cycle and induces apoptosis in different lymphoid cell types (Allione et al., 1995; Perillo et al., 1995; Walzel et al., 1997; Fouillit et al., 1998); (3) GAL1 dysregulates CD45-induced signaling, and as a result, Lyn kinase in BL cells (the present work). Future investigations of the mechanisms responsible for GAL1 regulation of tyrosine phosphatases and kinases will provide the crucial information necessary to link these observations. Nevertheless, there are sufficient data to suggest that some of these regulatory mechanisms may be analogous to the mechanisms deduced from the dimerization of CD45 by exogenous ligands (Weiss and Schlessinger, 1998). These observations suggest that CD45 function may be negatively regulated by its physiological ligands (Desai et al., 1993). Ligand-induced CD45 dimerization might block the catalytic site in CD45, thereby blocking substrate accessibility and inhibiting tyrosine phosphatase activity (Weiss and Schlessinger, 1998). In B cells the effects of CD45 dimerization may be mediated, at least in part, via control of Lyn activity. In this context, the results presented here provide evidence for the involvement of a signal transduction pathway, initiated through the inhibition of the tyrosine kinase activity of Lyn, in the cellular response to GAL1 in BL cells. Moreover, the observed posttranslational modification of Lyn is among the most rapid responses known for GAL1. This is in favor of the down-modulation of Lyn kinase, already activated in dividing cells, as a initiating step for GAL1 signal transduction. Lyn may induce tyrosine phosphorylation of a number of potent signaling proteins that have been shown to play vital roles in mediating complex biochemical pathways which contribute to the regulation of cell growth and development (Justement et al., 1994; Ogimoto et al., 1994; Greer and Justement, 1999). Therefore, one may suggest that although initial reports indicated that CD45/GAL1 interactions may induce apoptosis, this may not always be the case in B cells, as previously pointed out for T cells (Vespa et al., 1999). It is therefore likely that the function of GAL1 is not unequivocally that of a pro-apoptotic factor, but that it acts in a manner to regulate specific signal transduction processes, and that its effect may be, therefore, determined by the cell type and by the state of cell differentiation. |
Materials and methods |
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Cells
Two Epstein-Barr virus (EBV) negative BL cell lines, BL31, from Pr. Lenoir (CIRC, Lyon, France), and DG75, and the EBV+ BL cell line BL36 from Pr. Lenoir, were maintained in RPMI 1640 supplemented with 10% calf serum. Cultures were set up in triplicate. Viable cell numbers were determined by the MTS assay (Buttke et al., 1993
), using the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay kit (Promega). For viability assays, cells cultured for 24 h were treated with either 50 µl of GAL1 (700 nM) or 50 µl 0.15 M sterile NaCl. The proportion of living cells in cultures treated with GAL1 vs. the proportion in untreated cultures was calculated as follows: % cell viability = A490 (cells + GAL1)/A490 (cells + NaCl) x 100. Loss of membrane asymmetry was detected with merocyanin-540 (Sigma). Cells (5·105) were suspended in 100 µl of PBS, 0.1% BSA containing 5 µg/ml merocyanin. The samples were incubated for 3 min at room temperature and resuspended in 400 µl of PBS, and fluorescence was detected at 575 nm.
Antibodies and reagents
The anti-CD45 monoclonal antibody (mAb) was murine mAb T2/48 (Monostori et al., 1994; Oravecz et al., 1994). The polyclonal Ab against Lyn and the antiphosphotyrosine mAb 4G10 were purchased from Upstate Biotechnology Inc., Lake Placid, NY. Enolase was obtained from Sigma.
GAL1 purification
Human GAL1 was expressed in Escherichia coli and purified on a lactosyl-divinylsulfone-agarose column, essentially as described previously (Fouillit et al., 1998). Cells were extracted with ice-cold extraction buffer (50 mM Tris–HCl pH 7.4, 20 mM EDTA, 150 mM NaCl, 4 mM ß-mercaptoethanol, 0.25 mM phenylmethyl sulfonylfluoride (PMSF), 0.1 µM aprotinin, 1 µM pepstatin, 1 µM leupeptin, 1% Nonidet P-40), and the soluble extract was dialyzed against 50 mM Tris–HCl pH 7.4 containing 4 mM ß-mercaptoethanol, 0.25 mM PMSF, 0.02% sodium azide (MTB). The purified GAL1 was dialyzed against MTB and stored at –20°C. Just before the viability assays, the buffer was changed in a PD10 column equilibrated with sterile and apyrogenic 0.15 M NaCl solution.
Flow-cytometric analysis
Cells were suspended in PBS supplemented with 2% BSA, and incubated with biotinylated GAL1 (GAL1-biot) (Avellana-Adalid et al., 1990) for 30 min. The cells were then washed, and streptavidin-FITC was added at a final concentration of 2.5 µg/ml. They were incubated for another 30 min and washed twice, and cold paraformaldehyde (1% in PBS) was added. Cells were then suspended for analysis in a flow cytometer. In some experiments, the cells were incubated for 15 min with 25 mM thiodigalactoside in PBS after their incubation with GAL1-biot.
For antibody inhibition assay, biotinylated GAL1 (final concentration 2.3 µM) in PBS, 0.1% BSA was incubated with BL36 cells (5·105 cells per tube), for 30 min at room temperature. After washing, cells were incubated with phycoerythrin-conjugated streptavidin for 20 min. Then, 20 µl of anti-CD45-FITC mAb (Immunotech) was added and incubated for 15 min at 4°C. Controls were performed in the absence of either anti-CD45 or GAL1, and with 50 mM thiodigalactoside instead of anti-CD45
GAL1 and anti-CD45 blotting of membrane proteins
Washed cells were extracted in lysis buffer (20 mM Tris pH 7.4, 10 mM EDTA, 65 mM dithiothreitol, 0.1 µM aprotinin, 1 µM pepstatin, 1 µM leupeptin, and 30 mM lactose). After centrifugation, the pellet containing the cell membranes was solubilized with the same buffer containing 1% Nonidet P-40. The soluble supernatants were separated by SDS–PAGE and transferred to an Immobilon-P membrane (Millipore). Blots were incubated with anti-CD45 mAb (10 µg/ml), or GAL1-biot (10 µg/ml) for 2 h at room temperature. The blots were visualized by incubation with goat anti-mouse HRP-conjugated Ig diluted 1:20,000, or with streptavidin-HRP (Strep-HRP, 0.13 µg/ml), for 1 h at room temperature. Peroxidase activity was detected with PBS containing 0.2% H2O2 and 0.5 mg/ml diaminobenzidine.
Characterization of GAL1 receptors
GAL1 receptors were purified by a method adapted from Cosma (Cosma, 1997). GAL1 was immobilized on agarose beads (Cornillot et al., 1992), and equilibrated in MTB containing 1% Nonidet P-40. Cell lysate membrane glycoproteins were adsorbed by incubating 300 µl lysate (3.5 mg/ml) with 200 µl agarose-GAL1, for 3 h at room temperature. To remove unbound components, the agarose-GAL1-glycoprotein complex was washed extensively, first by MTB containing 0.25% Nonidet P-40 (MTB-P), and then by 0.1 M borate buffer pH 8.5, 0.5 M NaCl. The complex was then resuspended 1:4 in the borate buffer, incubated with NHS-biotin (100 µl, 1 mg/ml) for 30 min with gentle rotation and extensively washed with MTB-P to remove unreacted biotin. Biotinylated glycoproteins were eluted from agarose-GAL1 by incubation for 2 h at 4°C with 400 µl of MTB-P containing 0.15 M NaCl and the competitive inhibitor lactose (0.2 M), with rotation.
An aliquot of the eluate was then immunodepleted in CD45, using anti-CD45 mAb coupled to UltraLink Biosupport Medium (Pierce) according to the instructions of the manufacturer; 200 µl of the eluate was mixed with immobilized CD45, and the mixture was incubated overnight at 4°C. The immunoprecipitate was washed with immunoprecipitation buffer (50 mM sodium acetate pH 5.0, 0.5 M NaCl, 0.1% SDS, 1% Nonidet P-40, and 0.02% sodium azide) containing 0.2% Nonidet P-40, centrifuged to pellet the beads, and the beads were boiled in SDS sample buffer.
Finally, the different protein fractions were resolved by 8.5% SDS–PAGE, transferred to an Immobilon-P membrane, and detected either by incubation with anti-CD45 mAb (10 µg/ml) using ECL, or by probing with streptavidin–horseradish peroxidase (Strep-HRP, 0.13 µg/ml) complex. The peroxidase activity was visualized with the Opti-4CN kit (Bio-Rad). The image of the membranes was acquired from GS-700 Densitometer, and analyzed with Molecular Analyst Software (Bio-Rad).
Phosphotyrosine phosphatase assay
Assays of phosphotyrosine phosphatase (PTPase) were performed on BL36 membranes suspended in 100 mM sodium acetate, pH 6.0, 1 mM EDTA. PTPase activity was assayed against the phosphotyrosine analog p-nitro-phenyl-phosphate (10 mM) at room temperature by following the release of para-nitro-phenyl at 410 nm.
Tyrosine phosphorylation detection and in vitro kinase assay
BL36 cells were stimulated at a density of 4.105 with GAL1 (700 nM) for indicated times, lysed by addition of lysis buffer (50 mM Tris, pH 7.4, 10 mM EDTA, 4 mM ß-mercaptoethanol, protease inhibitors, 10 mM CHAPS, 1 mM sodium orthovanadate). After centrifugation at 20,000 x g for 30 min to remove insoluble material, lysates were incubated with UltraLink Immobilized Protein G (Pierce) coupled with Ab against Lyn. After incubation, immunoprecipitates were washed with immunoprecipitation buffer containing 0.2% Nonidet P-40. Samples were centrifuged to pellet the beads, and the beads were boiled in SDS sample buffer. Western blots with an equal load in each well were performed using the antiphosphotyrosine Ab 4G10.
For in vitro kinase assay, anti-Lyn immunoprecipitates were subjected to the reaction in kinase buffer (500 mM HEPES, pH 7.0, 10 MgCl2, 5 MnCl2, and 50 mM sodium orthovanadate, and 5 µM ATP) containing acid-treated enolase as an exogenous substrate, and resolved by SDS–PAGE. The phosphorylated substrate was visualized by Western blot using the antiphosphotyrosine Ab (Lankar et al., 1998).
Bidimensional electrophoresis of immunoprecipitates
Anti-Lyn Ab was coupled to UltraLink Biosupport Medium according to the instruction of the manufacturer (10 µl of Ab/10 mg of UltraLink Biosupport, per sample) in 0.6 M sodium citrate, 0.1 M sodium carbonate, pH 9. For each experimental condition 200 µl of cell lysate was incubated with a sample of support, immunoprecipitated, and then eluted with 500 µl of 7 M urea, 2 M thiourea, 2% (w/v) CHAPS, 65 mM DTT, 0.6% (v/v) IEF Buffer 4–7, and traces of bromophenol blue. Just prior use, the same buffer was added to the sample homogenate to obtain a final volume of 700 µl. Bidimensional electrophoresis were performed as described previously (Joubert-Caron et al., 1999). Briefly, the samples (350 µl per strip) were separated on the IPGphor unit platform (Amersham Pharmacia Biotech) at 19°C, and the separation was achieved with a total of 42,000 Vhs. After IEF, the strips were equilibrated for 15 min in 50 mM Tris–HCl, pH 8.6 containing 6 M urea, 1% (w/v) SDS, 65 mM DTT, 30% (v/v) glycerol, and trace of bromophenol blue, and 15 min with iodoacetamide (53 mM) instead of DTT. Excel gels 12–14% (24 cm wide, 18 cm long) were used for the second dimension electrophoresis. Silver-stained gels were scanned using a GS-700 densitometer, and the computer image analysis was carried out using MELANIE II software release 2.2 (Bio-Rad), allowing automatic quantification of protein spots, as well as the matching between our different bidimensional electrophoresis (Appel et al., 1997). The quantification of each spot of interest was expressed as percent volume (% V), where % V = spot volume /
volumes of all spots of interest.
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Acknowledgments |
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This work was supported by grants from the Ministère de l’Education Nationale de la Recherche et de la Technologie (MENRT), and from the Ligue Française contre le Cancer (Comité de Seine Saint-Denis). We gratefully acknowledge the expert technical help from Mr. P.Bissières (Service d’Hématologie Biologique, Bobigny). M.F. is a recipient of a MENRT fellowship from the French government.
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Abbreviations |
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TopAbstractIntroductionResults and discussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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BL, Burkitt lymphoma; EBV, Epstein-Barr virus; ECL, enhanced chemiluminescence; GAL1, galectin-1; mAb, monoclonal Ab; MTB, Tris buffer containing 4 mM ß-mercaptoethanol; MTB-P, MTB containing 0.25% Nonidet P-40; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxylethoxyphenyl)-2-(4-sulfophenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; PMSF, phenylmethyl sulfonylfluoride; PTPase, phosphotyrosine phosphatase; Strep-HRP, streptavidin–horseradish peroxidase.
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Footnotes |
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1 To whom correspondence should be addressed at: Laboratoire de Biochimie des Protéines et Protéomique, BCHL, UFR Léonard de Vinci, Université Paris 13, 74 rue Marcel Cachin, 93017 Bobigny, France
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