Molecular Composition of Staufen2-Containing Ribonucleoproteins in Embryonic Rat Brain

Messenger ribonucleoprotein particles (mRNPs) are used to transport mRNAs along neuronal dendrites to their site of translation. Numerous mRNA-binding and regulatory proteins within mRNPs finely regulate the fate of bound-mRNAs. Their specific combination defines different types of mRNPs that in turn are related to specific synaptic functions. One of these mRNA-binding proteins, Staufen2 (Stau2), was shown to transport dendritic mRNAs along microtubules. Its knockdown expression in neurons was shown to change spine morphology and synaptic functions. To further understand the molecular mechanisms by which Stau2 modulates synaptic function in neurons, it is important to identify and characterize protein co-factors that regulate the fate of Stau2-containing mRNPs. To this end, a proteomic approach was used to identify co-immunoprecipitated proteins in Staufen2-containing mRNPs isolated from embryonic rat brains. The proteomic approach identified mRNA-binding proteins (PABPC1, hnRNP H1, YB1 and hsc70), proteins of the cytoskeleton (α- and β-tubulin) and RUFY3 a poorly characterized protein. While PABPC1 and YB1 associate with Stau2-containing mRNPs through RNAs, hsc70 is directly bound to Stau2 and this interaction is regulated by ATP. PABPC1 and YB1 proteins formed puncta in dendrites of embryonic rat hippocampal neurons. However, they poorly co-localized with Stau2 in the large dendritic complexes suggesting that they are rather components of Stau2-containing mRNA particles. All together, these results represent a further step in the characterization of Stau2-containing mRNPs in neurons and provide new tools to study and understand how Stau2-containing mRNPs are transported, translationally silenced during transport and/or locally expressed according to cell needs.


Introduction
In neurons, mRNA transport is widely used to differentially regulate protein content in domains distant from the cell body [1]. Especially, mRNA transport and local translation are known to be involved in neuron development, synaptic functions and plasticity [2,3,4]. A current model stipulates that, along the way from nuclear export to dendritic anchoring, proteins are added or removed from the mRNP complexes in a dynamic way. It was proposed that these proteins finely control the successive steps that ensure proper expression of mRNA at specific times and space. Several combinations of mRNAs and proteins form a highly heterogeneous population of ribonucleoprotein (RNP) complexes that are linked to different forms of synaptic activity and/or plasticity [5,6,7]. In particular, two large families of RNPs have been suggested: mRNA particles and mRNA granules [3,6]. mRNA particles are distinguished from mRNA granules by the absence of ribosomes. It was suggested that RNA particles might represent the observed transport mRNPs [2]. Staufen2 (Stau2), a protein mainly expressed in brain is a well accepted player for mRNA localization [7,8]. The Stau2 gene expresses four protein isoforms of 62, 59, 56 and 52 kDa that are generated by differential splicing (Fig. 1) [7]. Stau2 binds doublestranded RNAs and is incorporated into mRNPs that move along microtubules in neuronal dendrites [6,7,8]. Interestingly, its level of expression in dendrites regulates the level of transported mRNAs showing the importance of Stau2 for mRNA transport. Likely as a consequence, neurons in which Stau2 has been downregulated by RNAi show a reduced density of dendritic spines, associated with a change in their morphology. These phenotypes result in reduced amplitude of the miniature excitatory postsynaptic currents, a measure of synaptic transmission [9].
Stau2 was described in both the nucleus and somatodendritic compartment of the cells [7,10]. Accordingly, Stau2 was shown to associate with nuclear factors suggesting an early role for Stau2 in mRNP assembly [11,12]. In the somatodendritic compartment, Stau2 associates with both mRNA granules and mRNA particles. On a sucrose gradient, Stau2 62 co-fractionates with ribosome-free particles whereas Stau2 59 and Stau2 52 were found in fractions that contained ribosomes [7]. However, the composition of these complexes is still largely unknown. In the work described in this paper, we immunoprecipitated Stau2-containing mRNPs and used a proteomic approach to identify Stau2-associated proteins. Several RNA-binding proteins and proteins of the cytoskeleton have been identified.

Isolation and characterization of Stau2-containing mRNPs
In order to identify the protein content of Stau2-containing RNPs, extracts of embryonic rat brains were prepared and endogenous Stau2 was immunoprecipitated using the polyclonal anti-Stau2 antibody L1 and its pre-immune serum as control ( Fig. 1). Following separation of co-immunoprecipitated proteins by SDS-PAGE, the gel was cut into bands of 3 mm and the proteins digested in-gel with trypsin. Resulting peptides were identified by mass spectrometry. In addition to Stau2, seven proteins were present in the Stau2 immunoprecipitate (Table 1 and Supplemental Table S1). Y box-binding protein 1 (YB1), polyadenylate-binding protein cytoplasmic 1 (PABPC1), heatshock cognate protein 70 (hsc70) and heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1) are RNA-binding proteins previously associated with other mRNPs, aand b-tubulin are protein components of the cytoskeleton and RUFY3 (rap2interacting protein X) is still poorly characterized. PABPC1, YB1, and hsc70 co-immunoprecipitate with Stau2 62 and Stau2 59 It was previously shown that Stau2 62 co-fractionated with ribosome-free mRNPs whereas Stau2 59 and Stau2 52 isoforms were found in heavy fractions that also contained ribosomes [7]. Therefore, we first tested whether the association between Stau2 and the identified RNA-binding proteins is specific for one Stau2 isoform or present in different complexes. In addition, we determined whether the association is direct or whether it involves an RNA intermediate. To this end, N2A cells were transfected with plasmids coding for Stau2 62 -HA 3 or Stau2 59 -HA 3 and tagged proteins as indicated (Fig. 2). Cell extracts were prepared and Stau2 was immunoprecipitated using anti-HA antibody, in the presence or absence of the micrococcal nuclease. Co-immunoprecipitated proteins were analyzed by western blotting using antimyc or anti-GFP antibody as indicated. In the absence of micrococcal nuclease, PABPC1-myc, YB1-CFP and hsc70-CFP were found in Stau2 62 -HA 3 and Stau2 59 -HA 3 immunoprecipitates indicating that they are present in the same complexes as Stau2-HA 3 isoforms (Fig. 2 A-C). In contrast, hnRNP H1-myc was not detected (Fig. 2D). When micrococcal nuclease was added to the cell extracts before immunoprecipitation, Stau2 62 -HA 3 and Stau2 59 -HA 3 interaction with hsc70-CFP was still observed (Fig. 2C) whereas interactions with PABPC1-myc and YB1-CFP were lost (Fig. 2 A,B). These results show that several RNAbinding proteins are present in the same complexes as Stau2 isoforms and suggest that only the Stau2/hsc70 interaction involved direct protein-protein interaction.

Direct interaction between hsc70 and Stau2 62
To confirm the interaction between hsc70 and Stau2 at the protein level, we performed two in vitro binding assays, GST-pull down and surface plasmon resonance (SPR). To this end, bacterially expressed GST, maltose-binding protein (MBP), GST-hsc70 and MBP-Stau2 62 fusion proteins were affinity purified (Fig. 3A). For the pull down assay, GST as a negative control and GST-hsc70 were attached to glutathione columns. Black, grey and white boxes represent double-stranded RNA-binding (dsRBD) consensus sequence having full, partial or no RNA-binding activity, respectively. Hatched boxes represent the tubulin-binding domain (TBD). (B) Immunoprecipitation of Stau2 isoforms from embryonic E17-18 rat brain extracts using two different polyclonal anti-Stau2 antibodies, L1 (St2-L1) and L2 (St2-L2). The specificity of these antibodies was previously reported [7]. Pre-immune (PI) sera were used as controls. The Stau2 56 isoform is not visible in these cell extracts. * represents a non-specific IgG band. doi:10.1371/journal.pone.0011350.g001 Their ability to bind MBP-Stau2 62 in the presence or absence of RNase A was tested by western blotting. In contrast to GST that failed to pull-down MBP-Stau2 62 , the GST-hsc70 fusion protein was able to bring down MBP-Stau2 62 even in the presence of RNase A (Fig. 3B). Similarly, MBP and MBP-Stau2 62 were fixed to the SPR sensor chip surface. Then GST and GTS-hsc70 were flowed over the SPR chip surface and their interaction with the immobilized proteins was monitored in real time. The resulting sensorgrams indicate that a direct interaction occurred between GST-hsc70 and MBP-Stau2 62 even in the presence of RNase A (Fig. 3C). These in vitro binding studies confirm the ability of hsc70 to interact with Stau2, independent of RNA.
ATP modulates the interaction between hsc70 and Stau2 62 hsc70 contains two functional domains, an N-terminal ATPase domain that contains the ATP/ADP-binding site and a Cterminal peptide-binding domain that contains the substratebinding pocket [13]. Upon ATP binding and hydrolysis a conformational change is induced in the ATPase domain [14,15] that modifies the structure of the substrate binding domain and consequently modulates its ability to bind substrates. In its ATP bound form, hsc70 binds and releases substrates quickly, while, in the ADP bound state, substrate binding and release are slow [16,17]. Therefore, we next tested whether the Stau2 62 /hsc70 association is sensitive to the presence of ATP. To this end, the co-immunoprecipitation experiment (Fig. 4A) and the SPR experiment ( Fig. 4B) were reproduced in the presence of 10 mM ATP. In both cases, the presence of ATP almost completely abolished the Stau2 62 /hsc70 interaction.

Co-localisation of Stau2 isoforms with YB1 and PABPC1 in dendrites of hippocampal neurons
Altogether, our data suggest that we have isolated Stau2containing mRNA particles, a sub-population of all Stau2associated complexes in brains [6], and that these mRNPs contain YB1, PABPC1 and/or hsc70. To determine whether these proteins can also be detected in the large granule complexes that are visible in dendrites, embryonic hippocampal neurons were first transfected with plasmids coding for PABPC1-myc, YB1-CFP, hsc70-CFP or hnRNP H1-myc and fixed. Tagged-proteins and endogenous Stau2 were detected with anti-myc or anti-GFP and anti-Stau2 antibodies, respectively (Fig. 5). In addition to their presence in the cell bodies, PABPC1-myc ( Fig. 5A) and YB1-CFP ( Fig. 5B) can be found as puncta in dendrites (Fig. 5A). Only a very small fraction of these puncta also stained with antibodies that recognized endogenous Stau2. This suggests that PABPC1 and YB1 are not components of the large Stau2-containing complexes in dendrites. Similarly, hsc70-CFP was homogeneously distributed in the cell body and dendrites and did not form observable puncta (Fig. 5C). Therefore, it is suggested that its association with Stau2 may occur outside the large dendritic RNP complexes. Finally, hnRNP H1-myc was strictly nuclear (Fig. 5D), indicating that, if confirmed, Stau2/hnRNP H1 association would be restricted to the nucleus.
The reverse experiment was also done. Hippocampal neurons were transfected with Stau2 62 -HA 3 and its co-localization with endogenous PABPC1 (Fig. 6A) and YB1 (Fig. 6B) was analyzed with specific antibodies. In these conditions, PABPC1 partly colocalized with Stau2 62 -HA 3 whereas YB1 displayed only a weak co-localization. Altogether, our results suggest that PABPC1, YB1, hsc70 and hnRNP H1 may be mostly associated with Stau2 in small mRNPs and mainly absent in the large Stau2-containing mRNA granules in dendrites.

Discussion
Stau2 is an RNA-binding protein mainly expressed in brain [7] and its importance for mRNA transport in dendrites [8,18] and synaptic functions [9] has been established. As a further step aiming to define its molecular functions in neurons and the mechanisms of regulation of Stau2-mediated mRNA transport and translation, we determined the molecular composition of Stau2-containing mRNPs. The absence of ribosomal proteins in the proteomics suggests that Stau2-containing particles have been characterized. They mainly contain components of the microtubules and RNA-binding proteins. While PABPC1, YB1 and hsc70 are clearly associated with Stau2-mRNPs, the presence of hnRNP H1 in these complexes is still unclear. It is possible that hnRNP H1 only transiently interacts with Stau2 in the nucleus and/or that it specifically associates with Stau2 56 or Stau2 52 isoform(s). Alternatively, hnRNP H1 may be a false positive hit in the proteomics. However, hnRNP H1 has previously been identified in polysomefree poly(A)-bound mRNA complexes [19] and in embryonic RNA granules [20] suggesting that it is a component of at least some mRNPs.
Post-transcriptional regulation of gene expression relies on a highly heterogeneous population of mRNP particles that ensure proper mRNA processing during splicing, nuclear trafficking, cytoplasmic localization, translation and/or decay [21,22]. The differential presence/absence of RNA-binding proteins and other cofactors in each mRNP determines the roles of each mRNP complex in cellular functions and the fate of associated mRNAs. They may also form organized domains such as nuclear speckles, P-bodies or stress granules that are visible under the microscope when using specific markers [23,24,25]. In dendrites of neurons, large ribonucleoprotein complexes shown to be associated with membranes and/or ribosomes can also be observed [2,3,6]. Our results at the biochemical and cellular levels are consistent with the possibility that we have isolated and characterized Stau2containing ribosome-free mRNPs. A minor population of soluble, ribosome-free Stau2-containing complexes was already described in neurons [6,7]. This population contained all differentially spliced Stau2 isoforms and was enriched with RNAs [6]. Accordingly, in a parallel approach, we also isolated mRNAs from the immunoprecipitated Stau2-containing mRNPs indicating that the proteomically identified proteins are components of complexes that also contain mRNAs [26]. The role of Stau2 and of its protein partners on the fate of associated mRNAs is still unclear. The presence in the proteomics of a nuclear protein (hnRNP H1), a protein and mRNA chaperone also involved in nuclear import/export (hsc70) and proteins that regulate translation initiation (PABPC1 and YB1) suggests that the isolated Stau2containning mRNPs may be those involved in mRNP formation in the nucleus and/or in post-transcriptional regulation of bound mRNAs (see below). Stau2 was shown to be involved in mRNA transport in cellular processes [6,8,18,27], and based on known functions of its paralog Stau1, it might also be implicated in the control of mRNA stability or translation [28,29].
Interestingly all Stau2-associated proteins except RUFY3 were previously described in different mRNPs. Indeed, aand btubulin, YB1, PABPC1, hsc70 and hnRNP H1, as well as Stau2, were all present in the heterogeneous populations of mRNA granules isolated from embryonic rat brains [20]. Different combinations of the proteins were also described in other mRNPs [19,30,31]. However, they are not universal components of all mRNPs since they were not identified in RNA granules isolated from post-natal rat brains [32] and only PABPC1 and tubulins were found in Stau1-containing mRNPs [33,34] suggesting that they play specialized roles for the transport and translation of specific mRNAs. In contrast, RUFY3 is specific to Stau2containing mRNPs. This poorly characterized protein is expressed in brain and peaks around post-natal day 4. It accumulates in growth cones of minor processes and axons. Down-regulation of RUFY3 expression by RNAi leads to an increase in the population of neurons bearing surplus axons [35]. Its molecular function and its role within RNPs are completely unknown.
One of the fundamental questions in the field is how mRNA translation is repressed during transport and reactivated in response to cell needs. It is believed that mRNA transport particles are translationally repressed at the level of initiation whereas ribosomeassociated granules are kept silent during elongation [3]. Accordingly, our proteomic results on Stau2-containing RNPs identified YB1 and PABPC1, two proteins known to modulate translation through interaction with initiation factors, as prominent candidates to fulfill translational regulation. YB1 is known to play key roles in cap-dependent mRNA stabilization [36] and translation [37,38]. It is viewed as a general translational repressor that maintains mRNPs in a translationally silent state by its ability to bind the 59cap structure thus displacing the initiation factors eIF4E and eIF4G from these mRNAs [36,37]. Interestingly, YB1 can be phosphorylated by the Akt kinase [39,40], the Rsk1/2 kinase and PKC alpha [41]. Its phosphorylation by the Akt kinase specifically diminishes its interaction with the capped 59 mRNA terminus reducing its ability  to inhibit cap-dependent translation. Therefore, upon activation of a signalling pathway, phosphorylation of YB1 would be an efficient mechanism to modulate local translation of Stau2-bound mRNAs in neurons. Similarly, PABPC1 is involved in both activation and repression of translation at the level of initiation. By binding simultaneously to the poly(A) tail of mRNAs and to the initiation factor eIF4G, PABPC1 facilitates the formation of the closed loop structure that facilitates translation. PABPC1 is also involved in translation inhibition by repressors [42]. Indeed, poly(A) tail, PABPC1 and eIF4G are all required to allow the translational inhibition of the ceruloplasmin transcript by the IFN-gammaactivated inhibitor of translation (GAIT) repressor. Similarly, PABPC1 is involved in the translational repression of its own transcript by binding an adenine-rich autoregulatory sequence (ARS) in the 59-untranslated region [43]. It also binds with lower affinities to a non-poly(A) cis-acting dendritic localizer sequence in the vasopressin mRNA [43,44,45]. In addition to their roles in translation, both YB1 [46] and PABPC1 [47] strongly bind tubulins. The association between YB1 and tubulin is believed to interfere with mRNA binding thus reducing the YB1/mRNA ratio and facilitating translation. Considering that Stau2 mRNPs are transported on microtubules in dendrites and that both aand btubulin were found in the proteomics, the presence of YB1 and PABPC1 in Stau2-containing mRNPs may contribute to the control of mRNA translation activation by regulating mRNA accessibility.
Another proteomically identified protein is hsc70. Hsc70 has a chaperone activity driven by cycles of ATP/ADP bound states [15]. It could be an important factor for Stau2 folding, thus enabling it to bind mRNAs and/or other protein partners. It may also more directly influence mRNA metabolism and/or translation through its ability to modulate the folding of mRNA [48] and to stabilize mRNA via its binding to AU-rich sequences [49]. Interestingly, its mRNA binding activity is inhibited by the binding of ATP molecules that compete for the same protein domain [48]. We show in this paper that ATP binding also inhibits its interaction with Stau2 (Fig. 4) suggesting that hsc70 through ATP binding may regulate the release of mRNAs and/or control the dissociation of Stau2-containing RNPs. Releasing and/or destabilising mRNAs at precise moments during synaptic activity can finely tune protein expression at synapses. Hsc70 can play two additional important roles in mRNP transport. First, hsc70 is known to regulate the nuclear export/import trafficking of several karyopherin family members [50,51]. Therefore, it could be an important factor for the nuclear shuttling of Stau2 and/or nuclear exit of Stau2-containing mRNPs. However, its role in the trafficking of exportin-5 and CRM1 which are known to export Stau2 from the nucleus [10,52] has not yet been tested. Finally, hsc70 is important for the release of the molecular motor kinesin from its vesicular cargo permitting its precise localization [53]. Stau2 mRNPs are transported on dendritic microtubules [7] and co-fractionate with tubulin and kinesin [6] making hsc70 an important candidate for the regulation of their transport. Altogether, these functions of hsc70, especially those related to protein and mRNA folding and to nuclear export/import, suggested that hsc70 is involved in mRNP formation and/or other early steps in the process of mRNA transport. This may explain why the presence of hsc70 in large Stau2-containing mRNA transport complexes in dendrites may not be required (Fig. 5).
In conclusion, our data suggest that we have characterized a heterogeneous population of Stau2-containing mRNPs from embryonic rat brain. Our study indicates that they are largely composed of proteins that are also components of other types of mRNPs. The biochemical characterization of Stau2-containing particles gives us new tools that will contribute to our understanding of mRNA transport in neurons and of its role in neurons.

Ethics statement
Pregnant Sprague-Dawley rats were purchased from Charles Rivers Canada. Our research involving animals has been conducted according to the guidelines of the Canadian Council of Animal Care (CCAC). Our project has been approved by the ''Comité de déontologie de l9expérimentation sur les animaux'' at the Université de Montréal.
Immunoprecipitation of Stau2-containing mRNPs was performed on cell extracts prepared from whole brains of E17-E18 rat embryos. Cells were dissociated with a manual putter and lysed in 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.5% Triton X-100, 15 mM EGTA, 1 mM DTT and complete EDTA-free protease inhibitor cocktail (Roche). Cell lysates were centrifuged at 9300 g for 10 min to remove nuclei and cell debris. After centrifugation, supernatants were incubated with a rabbit polyclonal anti-Stau2 antibody [7] for 2 hours at 4uC, then with a 50% protein Asepharose slurry for 2 hours at 4uC. Immune complexes were washed five times with the lysis buffer and eluted from the resin by heating at 95uC for 5 minutes in elution buffer (100 mM Tris-HCl pH 7.4, 200 mM DTT and 4% SDS). RNAs were isolated by Trizol (Invitrogen) extraction.
For the co-immunoprecipitation experiments, N2A cells were propagated in DMEM medium supplemented with 10% BSA serum (HyClone). Cells were co-transfected with plasmids coding for one of the following proteins: Stau2 59 -HA 3 or Stau2 62 -HA 3 [12] and either hsc70-CFP, YB1-CFP, hnRNP H1-myc or PABPC1-myc using the calcium-phosphate technique. Cells were collected 48 h post-transfection. Immunopurifications were performed as above using a mouse monoclonal anti-HA antibody. Detection of the co-immunoprecipitated proteins was done by western blotting with either rabbit polyclonal anti-HA (Sigma), goat anti-Myc (Bethyl) or goat anti-GFP (Rockland) antibodies. To asses if the interaction was RNA dependent, cell extracts were incubated for 30 min at room temperature with 300 U (300 U/ ml) of the Micrococcal Nuclease (Fermentas) in 1 mM CaCl2 before adding the antibody.

Proteomic techniques
Proteomic techniques were essentially done as before [20]. Briefly, immunoprecipitation eluates were separated by SDS-PAGE, stained with Coomasie Blue, and cut into 26 horizontal gel slices with each slice processed for in-gel trypsin digestion and peptide extraction. The extracted peptide mixtures were separated and analyzed in an automated system by nanoscale LC Q-TOF MS/MS. After fragmentation in the MS/MS mode, the resulting spectra were searched with Mascot (version 1.9.03; Matrix Science, London, UK) against a copy of the National Center for Biotechnology Information (NCBI) non-redundant protein database (June 21 st , 2004) restricted to the Mammalia taxonomy.

Protein expression and purification
Plasmids coding for GST-hsc70 and MBP-Stau2 were cloned into the pGEX (Amersham Pharmacia Biotech) and pMal-C (New England Biolabs) vectors, respectively. The fusion proteins were expressed in E. coli BL21 cells, following induction with IPTG for 2 h. Proteins were collected in PBS/1 mM DTT/1% Triton X-100/5 mM benzamidine at 4uC. GST-hsc70 and MBP-Stau2 proteins were purified on glutathione-Sepharose-4B and amylose affinity columns, respectively and eluted with 10 mM reduced glutathione in 50 mM tris-HCl (pH 8.0) or 10 mM of D-maltose in PBS, respectively. For some experiments, lysates were treated with 50 mg/ml RNAse A for 1 h at 4uC before column purification. Protein purification was monitored by SDS-PAGE. Proteins were detected by zinc staining and/or western blotting experiments.

GST-pull down assay
Bacterially expressed and purified GST and GST-hsc70 proteins were attached to glutathione columns. MBP-Stau2 or MBP were loaded onto the columns, extensively washed and eluted as recommended by the manufacturer. Eluted proteins were analyzed by western blotting using anti-GST and anti-MBP antibodies. For some experiments, protein extracts were treated with 50 mg/ml RNAse A for 1 h at 4uC before loading onto the columns.

Surface plasmon resonance (SPR) binding assay
Binding interactions between purified MBP-Stau2 and GST-hsc70 were examined in real time using a BIACORE 2000 instrument (GE Healthcare Bio-Sciences AB, Upssala, Sweden). Experiments were performed on research-grade CM5 sensor chip at 25uC using filtered (0.2 mm) and degassed HBS-EP [10 mM Hepes pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% (v/v) Surfactant P20]. Protein-grade detergents [10% (v/v) Tween-20, 10% (v/v) DDM] were from Calbiochem; all other chemicals were reagent grade quality. Immobilized sensor chip surfaces were prepared using the Biacore amine Coupling Kit. Briefly, 35 ml of a freshly mixed solution of 200 mM 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and 50 mM N-hydroxysuccinimide was injected (at a flow rate of 5 ml min 21 ) to activate surface-exposed carboxymethyl groups into reactive esters. Next, 120 ml of MBP-Stau2 diluted to 70 mg ml 21 in 10 mM sodium acetate pH 4.0 was injected (at a flow rate of 10 ml min 21 ) to generate amine-coupled protein surfaces. Finally, 70 ml of 1 M ethanolamine pH 8.5 was injected to deactivate excess reactive groups and remove any nonspecifically bound ligand. A reference surface was prepared in a similar manner with purified MBP. To test binding, purified GST-hsc70 (60 mg ml-1) or GST (negative control) were injected over the coupled surfaces at 10 ml min 21 (3 min association time and 2.5 min dissociation time). For all SPR assays, the surfaces were regenerated between sample injections at 50 ml min 21 with a 30 s single pulse of 0.4 M NaCl followed by a stabilization time after regeneration of 3 min. The assays were performed in duplicates with different batches of purified proteins.