Glycobiology Advance Access originally published online on September 21, 2005
Glycobiology 2006 16(1):22-28; doi:10.1093/glycob/cwj041
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Modulation of HSP70 GlcNAc-directed lectin activity by glucose availability and utilization
UMR 8576/CNRS, Glycobiologie Structurale et Fonctionnelle, IFR 118, Bâtiment C9, 59655 Villeneuve d’Ascq, France
1 To whom correspondence should be addressed; e-mail: tony.lefebvre{at}univ-lille1.fr
Received on May 4, 2005; revised on September 7, 2005; accepted on September 14, 2005
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Abstract |
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It is well-accepted that protein quality control (occurring either after protein synthesis or after cell damage) is mainly ensured by HSP, but the mechanism by which HSP decides whether the protein will be degraded or not is poorly understood. Within this framework, it has been hypothesized that O-GlcNAc, a cytosolic and nuclear-specific glycosylation whose functions remain unclear, could take a part in the protection of proteins against degradation by modifying both the proteins themselves and the proteasome. Because the synthesis of O-GlcNAc is tightly correlated to glucose metabolism and Hsp70 was endowed with GlcNAc-binding property, we studied the relationship between GlcNAc-binding activity of both Hsp70 and Hsc70 (the nucleocytoplasmic forms of HSP70 family) and glucose availability and utilization. We thus demonstrated that low glucose concentration, inhibition of glucose utilization with 2DG, or inhibition of glucose transport with CytB led to an increase of Hsp70 and Hsc70 lectin activities. Interestingly, the response of Hsp70 and Hsc70 lectin activities toward variations of glucose concentration appeared different: Hsp70 lost its lectin activity when glucose concentration was >5 mM (i.e., physiological glucose concentration) in contrast to Hsc70 that exhibited a maximal lectin activity for glucose concentration ~5 mM and at high glucose concentrations. This work also demonstrates that HSP70 does not regulate its GlcNAc-binding properties through its own O-GlcNAc glycosylation.
Key words: glucose / heat shock proteins / hexosamine biosynthetic pathway / lectin / O-GlcNAc
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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Members of the 70-kDa heat shock proteins family (HSP70) have been demonstrated to be GlcNAc-binding lectins (Lefebvre et al., 2001
; Guinez et al., 2004). The new concept of lectin-chaperone has arisen from growing evidences that the cytosolic and nuclear-specific glycosylation O-linked N-acetylglucosamine (O-GlcNAc) could act as a protective signal against proteasomal degradation. Beyond the myriad of proteins that are O-GlcNAc glycosylated, proteasome is itself modified with this atypical glycosylation (Sumegi et al., 2003; Zhang et al., 2003; Zachara and Hart, 2004) and moreover can be regulated by this posttranslational modification (Zhang et al., 2003; Zachara and Hart, 2004). O-GlcNAc was first described by Torres and Hart (1984) and, rapidly, it appeared that this glycosylation was enriched in the cytosolic and nuclear compartments and that it was not static (as classical N- and O-glycosylation) but highly dynamic. Even more, it can counteract the effect of phosphorylation at the same or at adjacent sites in a reciprocal manner (for review, see Kamemura and Hart, 2003). Despite these exciting features, roles of O-GlcNAc remain elusive. Nevertheless and as mentioned above, it appears probable that O-GlcNAc could act as a protective signal against proteasomal degradation. One of the precursor works in the field was that of Han and Kudlow (1997) showing that when cultured cells were glucose deprived or stimulated by cyclic-adenosine 5¢-monophosphate, the transcription factor Sp1 was hypoglycosylated leading to its rapid degradation. This degradation was sensitive to N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal (LLnL) and lactacystin suggesting a proteasomal degradation. Treatment of cells with glucosamine or glucose protected Sp1 against degradation. Later, it has been proposed that O-GlcNAc glycosylation of a site found within the PEST sequence of the beta-estrogen receptor could block protein degradation and thus play an opposite role to phosphorylation (in this context, it is well known that PEST sequence phosphorylation could activate degradation) (Cheng et al., 2000). More recently, the cadherins-to-cytoskeleton connecting protein plakoglobin has been shown to be O-GlcNAc modified near a destruction box (Hatsell et al., 2003).
Using a wide variety of stresses (hyperthermia, UVB, arsenite, ethanol, etc.), Zachara et al. (2004) demonstrated a quick increase of O-GlcNAc content in the treated cells. Furthermore, when the level of O-GlcNAc was increased by using PUGNAc (an O-GlcNAcase inhibitor) or by transfecting COS7 cells with O-linked N-acetylglucosamine transferase (OGT), the thermo-tolerance of cells was increased in contrast to a reduction or a blockade of O-GlcNAc resulting in an increase of the sensitivity of cells to stress.
A few years ago, it has been established that O-GlcNAc was intimately linked to glucose metabolism (for review, see Wells et al., 2003). About 2–5% of extracellular glucose could be used for O-GlcNAc modification of proteins through the hexosamine biosynthetic pathway (HBP). O-GlcNAc has been postulated to be a sensor implicated in insulin resistance and in the decrease of glucose uptake by cells. First, the hypothesis of a negative feedback of glucose transport regulation by the flux of glucose through HBP was suggested in insulin target cells (Marshall et al., 1991). Second, Robinson et al. (1993) have shown that when rat hemidiaphragms were incubated in glucosamine or in high glucose concentrations, glucose uptake decreased. Insulin-resistance effect and, consequently, glucose uptake decrease were correlated with a defect in GLUT4 plasma membrane glucose transporter (Cooksey et al., 1999). It has also been shown that glucosamine-induced insulin resistance was accompanied by an increase of UDP-GlcNAc concentration (Rossetti et al., 1995). Using transgenic overexpression of OGT, McClain et al. (2002) showed a type 2 diabetic phenotype. This observation suggested that insulin resistance and O-GlcNAc glycosylation are linked. In the same topic, incubation of rat epitrochlearis muscles with PUGNAc induced an increase in the O-GlcNAc level of proteins and a reduced glucose transport, suggesting that O-GlcNAc glycosylation of proteins can induce insulin resistance (Arias et al., 2004). Taken together, these observations strongly support the pivotal role of O-GlcNAc in reduced glucose transport and insulin resistance through HBP. In this field, it must be noted that numerous proteins involved in the metabolism of glucose are themselves O-GlcNAc modified: casein-kinase II, glycogen synthase-kinase-3 (Lubas and Hanover, 2000), and insulin-receptor substrate-1 and 2 (Patti et al., 1999).
Starting from the relation between glucose and O-GlcNAc and, since O-GlcNAc is a putative protector of proteins against proteasomal degradation, we previously demonstrated the existence of HSP70 lectin properties and we showed that 70-kDa heat shock protein (Hsp70), the cytosolic, and nuclear HSP70 induced form were endowed with a GlcNAc-specific lectin activity (Guinez et al., 2004). Intriguingly, this lectin activity increased with stress. We thus hypothesized that HSP70 lectin activity serves as protection of proteins via O-GlcNAc residues.
This article demonstrates the close relationship between HSP70 lectin properties and glucose status. Glucose deprivation, glucose transport inhibition, and inability to use glucose strongly modulate lectin activity. Against all expectations, modulation of the lectin property does not depend on a self-regulation of HSP70 by its own O-GlcNAc residues, because the addition of glucosamine in a glucose-depleted medium which restored the O-GlcNAc glycosylation of HSP70 did not abrogate its lectin activity. This last point was reinforced by the fact that O-GlcNAc deglycosylation of cell extract after beta-hexosaminidase treatment did not enhanced Hsp70 lectin property.
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Hsp70 and Hsc70 lectin activities are dependent upon glucose concentration
To test the hypothesis of a glucose concentration-dependent Hsp70/70-kDa heat shock cognate (Hsc70) lectin activity, HepG2 cells were grown on dishes in medium-containing various glucose concentrations. First, we used a broad range of glucose concentrations ranging from 0 to 100 mM. After cells lysis, proteins were run on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), electroblotted onto nitrocellulose, and stained with an anti-Hsp70 antibody either directly (ctrl) or after enrichment on GlcNAc-beads (GlcNAc-beads enr). After revelation by enhanced chemiluminescence (ECL) (Figure 1A, top panel), nitrocellulose sheet was stripped and stained with an anti-Hsc70 antibody (Figure 1A, bottom panel). The two chaperones possessed a lectin activity when cells were depleted in glucose (Glc, 0 mM), and when glucose concentration was increased to 12.5 mM, this lectin activity strongly decreased for both Hsp70 and Hsc70. On the other hand, when glucose concentration was increased from 12.5 to 100 mM, Hsc70 showed a lectin activity which progressively increased starting from 25 mM to be maximal at 75 mM of glucose, whereas Hsp70 did not show such an enhancement. Right panel of Figure 1A represents the O-GlcNAc pattern of the crude cellular protein extract for each glucose concentration condition. It should be pointed out that glycosylation was maximal between 50 and 100 mM of glucose. The same experiment was performed with moderate glucose concentrations, that is, between 0 and 12.5 mM (Figure 1B). Hsp70 showed a progressive decrease in its lectin activity up to 5 mM glucose (top panel) in contrast to Hsc70 (bottom panel) which showed a maximal activity at 5 mM (i.e., physiological glucose concentration). Figure 1B, right panel, represents an anti-O-GlcNAc antibody (RL2) staining of control extracts. A progressive increase in O-GlcNAc proteins content can be observed with glucose increase. These first experiments clearly correlate glucose concentration to lectin activity of HSP70 and underline that Hsp70 and Hsc70 lectin activities were expressed in a different manner according to the glucose concentration.
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Inhibition of glucose utilization or inhibition of glucose transport leads to an increase in Hsp70 lectin activity
To extend and reinforce the results described above and to strengthen the hypothesis that lectin activity of Hsp70 and Hsc70 are modulated by the glucose concentration, HepG2 cells were cultured either in presence of 2-deoxyglucose (2DG), a glucose analogue that perturbs utilization of glucose by competing interactions with proteins/enzymes using glucose as a substrate, or in presence of cytochalasin B (CytB), a glucose transporter inhibitor. Control experiments performed on total protein extracts showed a decrease in their O-GlcNAc content after staining with the anti-O-GlcNAc antibody (Figure 2A, for 2DG treatment, and Figure 2B, for CytB treatment, upper panels). In both cases, whatever the culture conditions (with/without glucose, with/without glucosamine, or at 42°C), Hsp70 and Hsc70 shared an increased GlcNAc-binding properties when 2DG or CytB were added to the culture medium (compare lanes 2 and 6 and lanes 10 and 14). These results showed that when glucose transport is inhibited by CytB or when glucose utilization was decreased by 2DG, GlcNAc lectin activities of HSP70 were enhanced, confirming the results presented in Figure 1 and demonstrating that lectin activities of Hsp70 and Hsc70 depend on glucose availability and utilization.
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Hsp70 and Hsc70 do not regulate their GlcNAc-binding properties with their own O-GlcNAc glycosylation
Previously reported data suggested an opposite relationship between the O-GlcNAc modification of Hsp70 and its capability to recognize exogenous O-GlcNAc residues (Guinez et al., 2004). In this report, we showed that in a glucose-free medium, Hsp70 was not O-GlcNAc modified but exhibited a high lectin activity toward O-GlcNAc, and in contrast in a normal culture medium (Dulbecco’s modified Eagle’s medium [DMEM] with 4.5 g L–1 of glucose) Hsp70 was glycosylated but was practically devoid of GlcNAc-binding property. Thus, we hypothesized that Hsp70 O-GlcNAc modification could occur on the lectin site avoiding subsequent binding on GlcNAc beads. To tentatively answer this question, we artificially increased O-GlcNAc level by incubating cells in a glucose-free medium supplemented with 5 mM glucosamine. As mentioned under Introduction, glucosamine can directly enter the HBP without the need of glutamine : fructose 6-phosphate amido-transferase (GFAT), the key- and rate-limiting enzyme of HBP. In these conditions, a lectin activity was induced both for Hsp70 (compared with glucose-deprived conditions but was lower to control, i.e., in presence of glucose) and for Hsc70 (the intensity of binding is more or less the same for the control and for the cell cultured in the presence of glucosamine), whereas the two chaperones were O-GlcNAc modified (Figure 3A, left panel for controls and right panel for GlcNAc-enriched and anti-O-GlcNAc antibody-enriched samples). This demonstrated that in these conditions, Hsp70 and Hsc70 lectin activities were dependent upon glucose deprivation and not dependent upon their own O-GlcNAc level. A second experiment invalidated this later hypothesis. HepG2 cells were exposed to a thermal stress for increasing periods (from 0 to 48 h). After cell lysis, GlcNAc-binding properties and glycosylation of Hsp70/Hsc70 were examined (Figure 3B). Both for Hsp70 and Hsc70, lectin properties and glycosylation reached maximal activities near 20 h: the time progress curves were similar for the lectin activity (left panel) and for the O-GlcNAc content (right panel) of Hsp70 and Hsc70. Figure 3C is a control of the O-GlcNAc glycosylation of total protein extract. This result reinforces the idea that Hsp70 and Hsc70 lectin properties are not regulated by their own O-GlcNAc modification. Finally, treatment of cell extracts with beta-hexosaminidase definitively confirmed these results, because after O-GlcNAc hydrolysis with beta-hexosaminidase treatment Hsp70 of cells cultured in normal conditions did not recover GlcNAc-binding property (Figure 4). Taken together, these results confirmed that HSP70 did not self-regulate their GlcNAc-binding properties with their own O-GlcNAc glycosylation.
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Discussion |
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The serine/threonine O-GlcNAc modification is widely expressed in cytosolic and nuclear compartments of eukaryotes. It modifies numerous proteins implicated in transcriptional processes (including transcription factors and RNA polymerase II) (Yang et al., 2002
), metabolic enzymes (Cieniewski-Bernard et al., 2004), nuclear pore proteins (Guinez et al., 2005), and many other proteins. Despite the great interest brought to the study of O-GlcNAc, the exact functions played by this simple glycosylation remain unknown. In this work, we focused our interest on the relationship between utilization of glucose and Hsp70/Hsc70 lectin activities. It has been demonstrated that a nonnegligible part of glucose that enters the cell was converted into UDP-GlcNAc (the donor of GlcNAc for OGT); the question arose to know whether cellular chaperones such as Hsp70 and Hsc70 were affected, particularly by their GlcNAc-binding activity, when glucose was limiting or in excess. To answer this question, HepG2 cells were cultured with increasing glucose concentrations ranging from 0 to 100 mM (4-fold the glucose concentration present in normal culture medium). Surprisingly, Hsp70 and Hsc70 lectin activities were differently affected (Figure 1A). Indeed, Hsp70 bound GlcNAc when cells were deprived in glucose, but lectin activity decreased after addition of glucose. In contrast, Hsc70 bound GlcNAc beads in glucose-deprived conditions but also for higher glucose concentrations (the optimal activity being between 75 and 100 mM of glucose). To be more representative of physiological conditions, lower glucose concentrations were used (from 0 to 12.5 mM, i.e., half-fold the glucose concentration of a normal culture medium). In these conditions, the lectin activities of Hsp70 and Hsc70 were different. In contrast to Hsp70 which bound GlcNAc when glucose concentration was <5 mM, Hsc70 reached a maximal binding activity at 5 mM. In both cases, the key glucose concentration was 5 mM, that is, the physiological glucose concentration. We thus demonstrated that the two chaperones did not work similarly, according to the glucose concentration. At low glucose concentration (<5 mM) owing to the stress, Hsp70 level and GlcNAc-binding activity are induced. When glucose concentration reaches a physiological or higher values (>5 mM), Hsp70 level decreases, and the lectin activity disappears in contrast to Hsc70 that shows an increasing lectin activity but at a constant protein expression level. Interestingly, the decrease of glucose concentration induced both a decrease in protein O-GlcNAc modification and an enhancement of Hsp70 lectin activity (Figure 1B, right panel); so a decrease in O-GlcNAc glycosylation could be compensated by a higher capacity of Hsp70 to recognize O-GlcNAc-modified cellular proteins.
To drive further this relationship between glucose and Hsp70/Hsc70 lectin activity, we used two drugs that either enabled glucose utilization or its transport into the cell. In both cases, even in normal conditions, lectin activity of Hsp70 and Hsc70 increased, showing the importance of glucose entry and utilization in the regulation of Hsp70/Hsc70 lectin properties (Figure 2A and B). One explanation for this phenomenon could be that when glucose deprivation occurs, the enhancement of HSP70 lectin activity observed counteracts the decrease of O-GlcNAc modification of cellular proteins. This could re-equilibrate the misbalance between O-GlcNAc and GlcNAc-binding properties.
The possibility that HSP70 lectin properties are regulated with HSP70 O-GlcNAc-glycosylation was tested. It appeared attractive that HSP70 could self-regulate their GlcNAc-binding properties with their own O-GlcNAc glycosylation. Three points came to invalidate this hypothesis. First, when HepG2 cells were cultured in absence of glucose but with glucosamine (to by-pass the rate-limiting enzyme of the HBP–GFAT—thus allowing the synthesis of UDP-GlcNAc and the transfer of O-GlcNAc residues), Hsp70 and Hsc70 were glycosylated and were endowed with GlcNAc-binding properties. In these conditions, it seems that glucose depletion was critical for activating GlcNAc properties of chaperones independently from their O-GlcNAc glycosylation. The second approach was to follow the GlcNAc-binding activity and the glycosylation progression of Hsp70 and Hsc70 during stress. The stress inflected to cells was a thermal one and not a glucose deprivation to maintain the formation of O-GlcNAc. In these conditions, we showed that the two features, that is, the O-GlcNAc glycosylation and the GlcNAc-binding property of both Hsp70 and Hsc70 evolved similarly along the stress period. This excludes an autoregulation of the chaperones with their own O-GlcNAc. Finally, after treatment of the total protein extract with beta-hexosaminidase, Hsp70 did not modify the lectin capacity. These observations indicate that the regulation of HSP70 lectin activity is mediated by something else than O-GlcNAc glycosylation. We can suppose that unidentified partners could modulate this property by interacting with chaperones. In this idea, the intervention of co-chaperones must be considered.
This article demonstrates the close relationship between the level of glucose and the lectin property of Hsp70 and Hsc70 toward GlcNAc residue. This phenomenon could be compared with the existing relationship between O-GlcNAc level and glucose concentration. The functions of such lectin activities are without any doubt in the protection of proteins against outer attacks. HSP are the guardian of the cell integrity, and this new function could be an additive weapon to carry their mission through.
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Materials and methods |
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Cell culture and treatments of cells
HepG2 cells were maintained in DMEM (Gibco, Cergy Pontoise, France) supplemented with 10% fetal calf serum (v/v), 2 mM L-glutamine, 5 IU/mL penicillin, and 50 µg/mL streptomycin at 37°C on a humidified atmosphere enriched with 5% CO2. Cultures were carried out on dishes (diameter 100 mm) preliminarily treated with 0.1% porcine gelatine (Sigma-Aldrich, Lyon, France).
Before stress, cells were washed with 10 mL of glucose-depleted medium and incubated either in this medium (for the starvation condition) or in this medium supplemented with glucose (cell culture tested, Sigma-Aldrich) at low (1–12.5 mM) or at high (25–100 mM) concentrations. Glucose-free medium was also supplemented with 5 mM glucosamine (cell culture tested, Sigma-Aldrich) for 24 h. 2DG (Sigma-Aldrich) was used at a concentration of 5 mM and CytB (Sigma-Aldrich) at a concentration of 0.1 mM. Thermal stress was induced by incubation of the cells for 24 h at 42°C in a 5% CO2-enriched atmosphere. For kinetic experiments, HepG2 were grown at 37°C and placed at 42°C from 0 to 48 h. Cell viability was determined by the Trypan blue exclusion method.
GlcNAc-binding proteins and O-GlcNAc-bearing proteins enrichment
HepG2 were first washed with 10 mL of cold phosphate-buffered saline (PBS, Gibco). Cells were lysed with a scrapper on ice either with a hypotonic buffer (10 mM Tris–HCl, 10 mM NaCl, 15 mM 2-mercaptoethanol, 1 mM MgCl2, and proteases inhibitors, pH 7.2) for lectin activity studies or with a detergent-containing buffer (DB) (10 mM Tris–HCl, 150 mM NaCl, 1% Triton X-100 (v/v), 0.5% sodium deoxycholate (w/v), 0.1% sodium dodecyl sulfate (w/v), and proteases inhibitors, pH 7.4) for the O-GlcNAc-content studies. Cellular extracts were centrifuged at 20,000 g for 30 min at 4°C. To test the lectin activity, supernatants were incubated with 30 µL of GlcNAc-coupled beads (N-acetyl-D-glucosamine immobilized on 6% beaded agarose with a spacer of five carbons, Sigma-Aldrich) at 4°C for 1 h. Beads were washed four times with binding buffer (20 mM Tris–HCl, 200 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and proteases inhibitor, pH 7.4). Specificity of binding has been tested with different sugar-coupled beads and with free sugar in excess (Guinez et al., 2004) and data not shown (glucose and GlcNAc). To study the O-GlcNAc glycosylation of proteins, immunoprecipitations with the anti-O-GlcNAc antibody (RL-2, Affinity Bioreagents, Golden, CO) were performed. RL-2 was added to a 1:250 final dilution, and cellular extracts were incubated at 4°C overnight. The bound proteins were then recovered after addition of protein G-Sepharose (Amersham Biosciences, Orsay, France) for 1 h at 4°C. Beads were gently centrifuged for 1 min and washed with the following buffers: DB; DB supplemented with 500 mM NaCl, DB/TNE (10 mM Tris–HCl, 150 mM NaCl, and 1 mM ethylenediaminetetraacetic acid [EDTA], pH 7.4) in equal volume, and finally with TNE alone.
Beta-hexosaminidase treatment
HepG2 extracts were adjusted to pH 5.2 with 100 mM acetate and incubated with Escherichia coli recombinant beta-hexosaminidase (Calbiochem, San Diego, CA) for 24 h at 37°C.
SDS–PAGE, western blotting, and antibody staining
Samples were analyzed by 10% SDS–PAGE under reducing conditions, and proteins were electroblotted onto nitrocellulose sheet (Amersham Biosciences). Membranes were first saturated for 45 min with 5% non-fatty acid milk in Tris-buffered saline (TBS)–Tween buffer (20 mM Tris–HCl, 150 mM NaCl, and 0.05% Tween [v/v], pH 8.0). Rabbit anti-Hsp70 polyclonal antibodies were incubated for 1 h at a dilution of 1:150,000 (Stressgen Bioreagents, Victoria, British Columbia). RL-2 anti-O-GlcNAc monoclonal antibodies were incubated overnight at 4°C at a dilution of 1:1000. Membranes were then washed three times with TBS–Tween for 10 min and incubated with either an anti-rabbit or an anti-mouse horseradish peroxidase-labeled secondary antibodies (Amersham Biosciences) at a dilution of 1:10,000 for 1 h. Three washes of 10 min each were performed with TBS–Tween, and the detection was carried out with ECL solution (Amersham Biosciences). Primary and secondary antibodies complexes were removed from the membranes with a stripping buffer (62.5 mM Tris–HCl, 2% SDS, 100 mM 2-mercaptoethanol, pH 6.5) for 30 min at 50°C, abundantly washed with TBS–Tween and reincubated with a rat anti-Hsc70 at a dilution of 1:1000 (Stressgen Bioreagents). Anti-rat secondary antibody labeled with horseradish peroxidase (Amersham Biosciences) was used at a dilution of 1:10,000 for 1 h. Polyclonal anti-beta-catenin was used at a dilution of 1:1000.
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C.G. is a recipient of a fellowship from the Ministère de la Recherche et de l’Enseignement. We thank le Centre National de la Recherche Scientifique and the University of Lille I. We are grateful to Dr. Anne Harduin-Lepers for the final reading of the manuscript and for English corrections.
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Abbreviations |
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2DG, 2-deoxyglucose; CytB, cytochalasin B; HBP, hexosamine biosynthetic pathway; Hsc70, 70-kDa heat shock cognate; Hsp70, 70-kDa heat shock protein; HSP70, members of the 70-kDa heat shock proteins family; O-GlcNAc, O-linked N-acetylglucosamine; OGT, O-linked N-acetylglucosamine transferase; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; TBS, Tris-buffered saline
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References |
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