New Arabidopsis thaliana Cytochrome c Partners: A Look Into the Elusive Role of Cytochrome c in Programmed Cell Death in Plants
2013; Elsevier BV; Volume: 12; Issue: 12 Linguagem: Inglês
10.1074/mcp.m113.030692
ISSN1535-9484
AutoresJonathan Martínez‐Fábregas, Irene Díaz‐Moreno, Katiuska González‐Arzola, Simon Janocha, José A. Navarro, Manuel Hervás, Rita Bernhardt, Antonio Díaz‐Quintana, Miguel Á. De la Rosa,
Tópico(s)Cell death mechanisms and regulation
ResumoProgrammed cell death is an event displayed by many different organisms along the evolutionary scale. In plants, programmed cell death is necessary for development and the hypersensitive response to stress or pathogenic infection. A common feature in programmed cell death across organisms is the translocation of cytochrome c from mitochondria to the cytosol. To better understand the role of cytochrome c in the onset of programmed cell death in plants, a proteomic approach was developed based on affinity chromatography and using Arabidopsis thaliana cytochrome c as bait. Using this approach, ten putative new cytochrome c partners were identified. Of these putative partners and as indicated by bimolecular fluorescence complementation, nine of them bind the heme protein in plant protoplasts and human cells as a heterologous system. The in vitro interaction between cytochrome c and such soluble cytochrome c-targets was further corroborated using surface plasmon resonance. Taken together, the results obtained in the study indicate that Arabidopsis thaliana cytochrome c interacts with several distinct proteins involved in protein folding, translational regulation, cell death, oxidative stress, DNA damage, energetic metabolism, and mRNA metabolism. Interestingly, some of these novel Arabidopsis thaliana cytochrome c-targets are closely related to those for Homo sapiens cytochrome c (Martínez-Fábregas et al., unpublished). These results indicate that the evolutionarily well-conserved cytosolic cytochrome c, appearing in organisms from plants to mammals, interacts with a wide range of targets on programmed cell death. The data have been deposited to the ProteomeXchange with identifier PXD000280. Programmed cell death is an event displayed by many different organisms along the evolutionary scale. In plants, programmed cell death is necessary for development and the hypersensitive response to stress or pathogenic infection. A common feature in programmed cell death across organisms is the translocation of cytochrome c from mitochondria to the cytosol. To better understand the role of cytochrome c in the onset of programmed cell death in plants, a proteomic approach was developed based on affinity chromatography and using Arabidopsis thaliana cytochrome c as bait. Using this approach, ten putative new cytochrome c partners were identified. Of these putative partners and as indicated by bimolecular fluorescence complementation, nine of them bind the heme protein in plant protoplasts and human cells as a heterologous system. The in vitro interaction between cytochrome c and such soluble cytochrome c-targets was further corroborated using surface plasmon resonance. Taken together, the results obtained in the study indicate that Arabidopsis thaliana cytochrome c interacts with several distinct proteins involved in protein folding, translational regulation, cell death, oxidative stress, DNA damage, energetic metabolism, and mRNA metabolism. Interestingly, some of these novel Arabidopsis thaliana cytochrome c-targets are closely related to those for Homo sapiens cytochrome c (Martínez-Fábregas et al., unpublished). These results indicate that the evolutionarily well-conserved cytosolic cytochrome c, appearing in organisms from plants to mammals, interacts with a wide range of targets on programmed cell death. The data have been deposited to the ProteomeXchange with identifier PXD000280. Programmed cell death (PCD) 1The abbreviations used are:PCDprogrammed cell deathCcCytochrome cApaf-1apoptosis protease-activating factor-1CED-4cell death abnormality-4DarkDrosophila Apaf-1-related killerBiFCBimolecular fluorescence complementationCPTCamptothecinSPRsurface plasmon resonanceNAAnaphthalene acetic acidMSMurashige and Skoog mediumTS-4BThiol-Sepharose 4BYFPyellow fluorescent proteinDAPI4′,6-diamidino-2-phenylindoleDMEMDulbecco's modified Eagle's medium. 1The abbreviations used are:PCDprogrammed cell deathCcCytochrome cApaf-1apoptosis protease-activating factor-1CED-4cell death abnormality-4DarkDrosophila Apaf-1-related killerBiFCBimolecular fluorescence complementationCPTCamptothecinSPRsurface plasmon resonanceNAAnaphthalene acetic acidMSMurashige and Skoog mediumTS-4BThiol-Sepharose 4BYFPyellow fluorescent proteinDAPI4′,6-diamidino-2-phenylindoleDMEMDulbecco's modified Eagle's medium. is a fundamental event for the development of multicellular organisms and the homeostasis of their tissues. It is an evolutionarily conserved mechanism present in organisms ranging from yeast to mammals (1Vander Heiden M.G. Chandel N.S. Williamson E.K. Schumacker P.T. Thompson C.B. Bcl-XL regulates the membrane potential and volume homeostasis of mitochondria.Cell. 1997; 91: 627-637Abstract Full Text Full Text PDF PubMed Scopus (1235) Google Scholar, 2Matsuyama S. Nouraini S. Reed J.C. Yeast as a tool for apoptosis research.Curr. Opin. Microbiol. 1999; 2: 618-623Crossref PubMed Scopus (54) Google Scholar, 3Sundström J.F. Vaculova A. Smertenko A.P. Savenkov E.I. Golovko A. Minina E. Tiwari B.S. Rodriguez-Nieto S. Zamyatnin A.A. Välineva T. Saarikettu J. Frilander M.J. Suarez M.F. Zavialov A. Stahl U. Hussey P.J. Silvennoinen O. Sundberg E. Zhivotovsky B. Bozhkov P.V. Tudor staphylococcal nuclease is an evolutionarily conserved component of the programmed cell death degradome.Nat. 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In mammals, cytochrome c (Cc) and dATP bind to apoptosis protease-activating factor-1 (Apaf-1) in the cytoplasm, a process leading to the formation of the Apaf-1/caspase-9 complex known as apoptosome. This apoptosome subsequently activates caspases-3 and -7 (4Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cytochrome c and dATP-dependent formation of Apaf-1/Caspase-9 complex initiates an apoptotic protease cascade.Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6219) Google Scholar, 5Yu X. Acehan D. Ménétret J.F. Booth C.R. Ludtke S.J. Riedl S.J. Shi Y. Wang X. Akey C.W. A structure of the human apoptosome at 12.8 Å resolution provides insights into this cell death platform.Structure. 2005; 13: 1725-1735Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). 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Thus, the participation of Cc in the onset and progression of PCD needs to be further elucidated. Even in the case of mammals, the role(s) of Cc in the cytoplasm during PCD remain(s) controversial. Recently, new putative functions of Cc, going beyond the already-established apoptosome assembly process, have been proposed in the nucleus (19Ruíz-Vela A. González de Buitrago G. Martínez-A C. Nuclear Apaf-1 and cytochrome c redistribution following stress-induced apoptosis.FEBS Lett. 2002; 517: 133-138Crossref PubMed Scopus (32) Google Scholar, 20Nur-E-Kamal A. Gross S.R. Pan Z. Balklava Z. Ma J. Liu L.F. Nuclear translocation of cytochrome c during apoptosis.J. Biol. Chem. 2004; 279: 24911-24914Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) and the endoplasmic reticulum (21Boehning D. Patterson R.L. Sedaghat L. Glebova N.O. Kurosaki T. Snyder S.H. Cytochrome c binds to inositol (1,4,5) trisphosphate receptors, amplifying calcium-dependent apoptosis.Nat. 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The multiple functions of cytochrome c and their regulation in life and death decisions of the mammalian cell: From respiration to apoptosis.Mitochondrion. 2011; 11: 369-381Crossref PubMed Scopus (371) Google Scholar), are as yet fully understood. This current state of affairs demands deeper exploration of the additional roles played by Cc in nonmammalian species. In this study, putative novel Cc-partners involved in plant PCD were identified. For this identification, a proteomic approach was employed based on affinity chromatography and using Cc as bait. The Cc-interacting proteins were identified using nano-liquid chromatography tandem mass spectrometry (NanoLC-MS/MS). These Cc-partners were then further confirmed in vivo through bimolecular fluorescence complementation (BiFC) in A. thaliana protoplasts and human HEK293T cells, as a heterologous system. Finally, the Cc-GLY2, Cc-NRP1 and Cc-TCL interactions were corroborated in vitro using surface plasmon resonance (SPR). These results indicate that Cc is able to interact with targets in the plant cell cytoplasm during PCD. Moreover, they provide new ways of understanding why Cc release is an evolutionarily well-conserved event, and allow us to propose Cc as a signaling messenger, which somehow controls different essential events during PCD. Plasmid pCytA (25Rodríguez-Roldán V. García-Heredia J.M. Navarro J.A. Hervás M. De la Cerda B. Molina-Heredia F.P. De la Rosa M.A. A comparative analysis of the reactivity of plant, horse, and human respiratory cytochrome c towards cytochrome c oxidase.Biochem. Biophys. Res. Commun. 2006; 346: 1108-1113Crossref PubMed Scopus (22) Google Scholar), containing the coding region for A. thaliana Cc, was used to obtain the Cc mutant A111C, in which the C-terminal alanine was replaced by a cysteine, through mutagenic PCR. The oligonucleotides designed to build the A111C mutant were 5′-gaaggcacctgttgatgaattc-3′ and 3′-cttccgtggacaactacttaag-5′. The A111C mutant was expressed and further purified using ionic exchange chromatography, a process previously described for wild-type Cc by Rodríguez-Roldán et al. (25Rodríguez-Roldán V. García-Heredia J.M. Navarro J.A. Hervás M. De la Cerda B. Molina-Heredia F.P. De la Rosa M.A. A comparative analysis of the reactivity of plant, horse, and human respiratory cytochrome c towards cytochrome c oxidase.Biochem. Biophys. Res. Commun. 2006; 346: 1108-1113Crossref PubMed Scopus (22) Google Scholar). A. thaliana MM2d cell suspension cultures (Bayer CropScience) were grown in 1 × Murashige and Skoog (MS) medium (Duchefa Biochemie) supplemented with 30 g/L sucrose (Sigma-Aldrich), 0.5 mg/L NAA (Sigma-Aldrich), 0.05 mg/L kinetin (Sigma-Aldrich), 200 mg/L cefotaxime (Duchefa Biochemie) and 200 mg/L penicillin (Duchefa Biochemie) at 100 rpm and 25 °C. PCD was then induced according to the procedure described by De Pinto et al. (26De Pinto M.C. Paradiso A. Leonetti P. De Gara L. Hydrogen peroxide, nitric oxide and cytosolic ascorbate peroxidase at the crossroad between defence and cell death.Plant J. 2006; 48: 784-795Crossref PubMed Scopus (174) Google Scholar). Explained briefly, a stationary phase culture was diluted 5:100 (v/v). Following 3 days of growth under normal conditions, 35 mm H2O2 was added to 100 ml cell suspension cultures. Cell viability was measured using the trypan blue dye exclusion test as described by De Pinto et al. (27De Pinto M.C. Francis D. De Gara L. The redox state of the ascorbate-dehydroascorbate pair as a specific sensor of cell division in tobacco BY-2 cells.Protoplasma. 1999; 209: 90-97Crossref PubMed Scopus (223) Google Scholar) and cells were counted with a hemocytometer. The MM2d cell viability rate was calculated dividing the number of viable cells by the total number of cells. Following the collection of MM2d cells through centrifugation at 1000 × g for 10 min, cell morphology was analyzed and visualized using an Olympus BX60 fluorescence microscope. Protein content was determined using the Bradford assay (28Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215632) Google Scholar), whereas chlorophyll determination carried out according to MacKinney's protocol (29MacKinney G. Absorption of light by chlorophyll solutions.J. Biol. Chem. 1941; 140: 315-322Abstract Full Text PDF Google Scholar). Cell extracts from 0.5 L of culture containing either untreated or 35 mm H2O2-treated cells were prepared for affinity chromatography purification. In both cases, cells were harvested following centrifugation at 1000 × g for 5 min, washed twice in PBS, pelleted again and resuspended to be further lysed by sonication in buffer I (50 mm Tris-HCl (pH 7.5), 50 mm NaCl, 0.25% Triton X-100) supplemented with 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg·ml−1 leupeptin and 10 μg/ml of soybean trypsin inhibitor. Cellular debris was then removed through centrifugation at 20,000 × g for 30 min at 4 °C. Protein aliquots were stored at −80 °C. As previously described in Azzi et al. (30Azzi A. Bill K. Broger C. Affinity chromatography purification of cytochrome c binding enzymes.Proc. Natl. Acad. Sci. U.S.A. 1982; 79: 2447-2450Crossref PubMed Scopus (27) Google Scholar), affinity chromatography was carried out in a column prepared by the covalent linkage of the Cc mutant A111C to the Thiol-Sepharose 4B (TS-4B) matrix (Pharmacia). As a control, a TS-4B matrix devoid of Cc (Blank TS-4B) was also prepared. MM2d cell extracts, both those untreated and treated with 35 mm H2O2, were loaded into the columns, both with and without Cc. The columns were washed with 30 ml of buffer I and 30 ml of buffer II (50 mm Tris-HCl (pH 7.5), 75 mm NaCl) to remove nonspecifically bound proteins. Proteins interacting with greater strength were then eluted with 30 ml of buffer III (50 mm Tris-HCl (pH 7.5), 300 mm NaCl), collected, lyophilized, and stored at −80 °C before being analyzed using NanoLC-MS/MS. Four sets of samples were thus obtained: (1) untreated cell extracts loaded into Blank TS-4B column, (2) untreated cell extracts loaded into the Cc TS-4B column, (3) cell extracts treated with H2O2 and purified with the Blank TS-4B column, and (4) cell extracts treated with H2O2 and purified using the Cc TS-4B column. Before the performance of NanoLC-MS/MS analysis, the purified protein samples above were digested with trypsin. Thus, samples were treated with 8 m urea and 10 mm dithiothreitol (DTT). Following 1 h of incubation at 37 °C, iodoacetamide was added until a final concentration of 55 mm was reached and incubated for 1 h in the dark at room temperature. The samples were diluted with ammonium bicarbonate 4× until obtaining a final concentration of 2 m urea. Finally, 25 mg of recombinant trypsin was added and the mixture was incubated overnight at 37 °C. The resulting peptides were analyzed using NanoLC-MS/MS on a linear trap quadrupole (LTQ; Thermo Electron), a linear ion trap mass spectrometer. The peptides were separated in a BioBasic C-18 PicoFrit column (75 μm (internal hamester) by 10 cm; New Objective) at a flow rate of 200 nL/min. Water and acetonitrile, both containing 0.1% formic acid, were used as solvents A and B, respectively. The peptides were trapped and desalted in the trap column for 5 min. The gradient was started and maintained for 5 min at 5% B, then ramped to 50% B over 120 min, ramped to 70% over 10 min and finally maintained at 95% B for another 10 min. The mass spectrometer was operated in data-dependent mode to automatically switch between full MS and MS/MS acquisition. Parameters for ion scanning were the following: full-scan MS (400–1800 m/z) plus top seven peaks Zoom/MS/MS (isolation width 2 m/z), normalized collision energy 35%. Peak lists from all MS/MS spectra were extracted from the Xcalibur RAW files using a freely available program DTAsupercharge v1.19 (http://msquant.sourceforge.net). For protein identification, the UniProt Arabidopsis protein database 100323 (90895 sequences, 33249465 residues) was searched using a local license for MASCOT 2.1. Database search parameters used were the following: trypsin as enzyme; peptide tolerance, 300 ppm; fragment ion tolerance, 0.6 Da; missed cleavage sites,1, and fixed modification, carbamidomethyl cysteine and variable modifications, methionine oxidation. In all protein identification, probability scores were greater than the score established by MASCOT (30Azzi A. Bill K. Broger C. Affinity chromatography purification of cytochrome c binding enzymes.Proc. Natl. Acad. Sci. U.S.A. 1982; 79: 2447-2450Crossref PubMed Scopus (27) Google Scholar) as significant, with a p value less than 0.05. The cDNA coding available for nine out of the 10 Cc potential targets previously identified by the proteomic approach were purchased (ABRC Stocks, The Ohio State University, Columbus, OH, USA). The cDNA of Cc and its novel protein partners were fused with the C-end fragment of the yellow fluorescent protein (cYFP) of the pSPYCE vector and with the N-end part of the YFP (nYFP) of the pSPYNE vector, respectively (31Walter M. Chaban C. Schütze K. Batistic O. Weckermann K. Näke C. Blazevic D. Grefen C. Schumacher K. Oecking C. Harter K. Kudla J. Visualization of protein interactions in living plants cells using bimolecular fluorescence complementation.Plant J. 2004; 40: 428-438Crossref PubMed Scopus (1269) Google Scholar). As a negative control, protoplasts were transfected with the chromatin-remodeling complex element SWI3B, which is unable to interact with Cc. The oligonucleotides indicated in the supplemental Data (supplemental Fig. S1A) were used to amplify the cDNAs while introducing proper restriction sites by PCR. In supplemental Fig. S1B, a scheme is shown of the vector constructs used for the BiFC assays. Similarly, supplemental Fig. S2A represents the oligonucleotides required for cloning cDNAs into YFP vectors. BiFC experiments in human HEK293T cells were assayed after cloning Cc cDNA into the cYFP vector and the cDNA of its targets into the nYFP vector (supplemental Fig. S2B) (32Gandia J. Galino J. Amaral O.B. Soriano A. Lluís C. Franco R. Ciruela F. Detection of higher-order G protein-coupled receptor oligomers by a combined BRET-BiFC technique.FEBS Lett. 2008; 582: 2979-2984Crossref PubMed Scopus (90) Google Scholar). As discussed by Hu et al. (33Hu C.D. Grinberg A.V. Kerppola T.K. Visualization of protein interactions in living cells using bimolecular fluorescence complementation (BiFC) analysis.Curr. Protoc. Cell Biol. 2006; (Chapter 21, Unit 21.3)PubMed Google Scholar), pBiFC-bJunYN155 and pBiFC-bFosYC155 were used as positive controls, whereas pBiFC-bJunYN155 and pBiFC-bFosΔZipYC155 were employed as negative controls. Protoplasts were generated from 1-week-old A. thaliana MM2d cell cultures grown in MS medium. A 50 ml aliquot of cells were collected using centrifugation at 1500 rpm for 5 min and then resuspended in 50 ml of MS-Glucose/Mannitol (0.34 m), cellulose 1% and macerozyme 0.2%. Cells were incubated in this buffer for 3 h at 50 rpm in the dark to facilitate the digestion of the cell wall. Resulting protoplasts were collected following two, 5-min rounds of centrifugation at 800 rpm with a wash with 25 ml of MS-Glucose/Mannitol (0.34 m) carried out between centrifugations. The final pellet was resuspended in MS-Sucrose (0.28 m) and centrifuged at 800 rpm for 5 min. The A. thaliana protoplasts were recovered from supernatant. Following Sheen's protocol (34Sheen J. Signal transduction in Maize and Arabidopsis Mesophyll protoplasts.Plant Physiol. 2001; 127: 1466-1475Crossref PubMed Scopus (527) Google Scholar), protoplasts were transiently transfected with the pSPYCE/pSPYNE BiFC vectors and incubated overnight; on PCD induction with 35 mm H2O2, the resulting fluorescence was monitored. HEK293T cells were grown in Dulbecco's modified Eagle's medium (DMEM; PAA, Etobicoke, ON) supplemented with 2 mm l-glutamine (Invitrogen, Carlsbad, CA), 100 U/ml streptomycin (Invitrogen), 100 μg/ml penicillin (Invitrogen) and 10% heat-inactivated fetal bovine serum (PAA) at 37 °C in a humidified atmosphere of 5% CO2/95% air. HEK293T cells were grown to 80% confluence in 24-well plates with 500 μl of DMEM, containing 20 mm coverslips. Cells were transfected with the YFP BiFC vectors using the Lipofectamine 2000 Transfection Reagent (Invitrogen) following the manufacturer's instructions. To favor the protein expression of both constructs, the transfected cells were then incubated for 24 h at 37 °C. Apoptosis was further induced with 10 μm CPT (camptothecin) for 6 h and in vivo binding was assessed through YFP reconstitution visualized with fluorescence microscopy. Nuclei were then stained with 4′,6-diamidino-2-phenylindole (DAPI). The HEK293T cells were harvested 48 h after transfection through centrifugation at 1500 rpm for 5 min. Total cell extracts were obtained through repeated freeze-thaw cycles. SDS-PAGE was performed using 12% polyacrylamide gels. Proteins were transferred onto nitrocellulose membranes (BioRad, Hercules, CA) using a semidry transfer system and immunoblotted with a rabbit anti-EGFP polyclonal antibody (1:1,000; Biovision Research Products). A horseradish-peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:12,000; Sigma-Aldrich) was then used for detection. The immunoreactive bands were developed using ECL Plus Western blotting Detection System (Amersham Biosciences). Wild-type A. thaliana Cc was cloned in the pBTR vector under lac promoter and expressed in E. coli BL-21. For this, 25 ml of precultures were grown overnight at 37 °C in LB medium. 2.5 ml of preculture was used to inoculate 2.5 L of the same medium in a 5 L Erlenmeyer flask. The culture was shaken at 30 °C for 24 h, after which further protein purification was carried out as indicated in Rodríguez-Roldán et al. (25Rodríguez-Roldán V. García-Heredia J.M. Navarro J.A. Hervás M. De la Cerda B. Molina-Heredia F.P. De la Rosa M.A. A comparative analysis of the reactivity of plant, horse, and human respiratory cytochrome c towards cytochrome c oxidase.Biochem. Biophys. Res. Commun. 2006; 346: 1108-1113Crossref PubMed Scopus (22) Google Scholar). Proteins interacting with A. thaliana Cc—GAPDC1, GLY2, NRP1, and TCL—were cloned in the pET-28a vector under the T7 promoter. cDNAs coding for Cc targets were purchased from ABRC. These constructs were used to express the Cc-targets in the E. coli BL-21 (DE3) RIL strain. 250 ml precultures in LB medium supplemented with 50 μg/ml kanamycin were grown overnight and used to inoculate 2.5 L of LB medium in 5 L flasks. Following the induction of cultures (1 mm IPTG) and growth at 30 °C for 24 h, cells were harvested at 6000 rpm for 10 min and resuspended in 40 ml lysis buffer (20 mm Tris-HCl buffer (pH 8), 0.8 m NaCl, 10 mm imidazole, 0.01% phenylmethylsulphonyl fluoride (PMSF), 0.2 mg/ml lysozyme, 5 mm DTT and 0.02 mg/ml DNase), sonicated for 4 min and then centrifuged at 20,000 rpm for 20 min. Proteins were further purified by means of an Ni-column (GE Healthcare). The formation of complexes between A. thaliana Cc and its protein partners—GAPDC1, GLY2, NRP1, and TCL—was assayed with SPR using a BiaCore 3000 and CM4 Chips. An automated desorption procedure was performed before each experiment to ensure the cleanliness of the BiaCore tubing, channels, and sample injection port. The initial electrostatic attraction of A. thaliana Cc to the CM4 Sensor Chip surface was assessed by taking into account its isoelectric point and was optimized to pH 5.8. The plant Cc was then covalently attached to the matrix using standard amine-coupling chemistry, as previously described (35Janocha S. Bichet A. Zöllner A. Bernhardt R. Substitution of lysine with glutamic acid at position 193 in bovine CYP11A1 significantly affects protein oligomerization and solubility but not enzymatic activity.Biochim. Biophys. Acta. 2011; 1814: 126-131Crossref PubMed Scopus (12) Google Scholar). A reference flow cell was used as a control in which the chip surface was treated as described above, but without the injection of plant Cc. The binding measurements were performed at 25 °C using HBS-EP buffer containing 10 mm HEPES, 150 mm NaCl, 3 mm EDTA, and 0.005% surfactant P20, adjusted to pH 7.4. Interactions between plant Cc and its protein partners were analyzed by flowing several GAPDC1, GLY2, NRP1 and TCL proteins at different concentrations (from 0.1 to 10 μm) over the Cc-modified surface at a flow rate of 10 μl/min. Each concentration was injected at least three times. In each sensogram, the signals from the reference flow cell surface
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