A Human Protein Atlas for Normal and Cancer Tissues Based on Antibody Proteomicsстатья из журнала
Аннотация: Antibody-based proteomics provides a powerful approach for the functional study of the human proteome involving the systematic generation of protein-specific affinity reagents. We used this strategy to construct a comprehensive, antibody-based protein atlas for expression and localization profiles in 48 normal human tissues and 20 different cancers. Here we report a new publicly available database containing, in the first version, ∼400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. Each image has been annotated by a certified pathologist to provide a knowledge base for functional studies and to allow queries about protein profiles in normal and disease tissues. Our results suggest it should be possible to extend this analysis to the majority of all human proteins thus providing a valuable tool for medical and biological research. Antibody-based proteomics provides a powerful approach for the functional study of the human proteome involving the systematic generation of protein-specific affinity reagents. We used this strategy to construct a comprehensive, antibody-based protein atlas for expression and localization profiles in 48 normal human tissues and 20 different cancers. Here we report a new publicly available database containing, in the first version, ∼400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. Each image has been annotated by a certified pathologist to provide a knowledge base for functional studies and to allow queries about protein profiles in normal and disease tissues. Our results suggest it should be possible to extend this analysis to the majority of all human proteins thus providing a valuable tool for medical and biological research. Antibody-based tissue profiling allows a streamlined approach for generating expression data both for normal and disease tissues (1Uhlen M. Ponten F. Antibody-based proteomics for human tissue profiling.Mol. Cell. Proteomics. 2005; 4: 384-393Google Scholar). It is also possible to generate protein expression data on many different individual patients to evaluate heterogeneity of tissue profiles (2Kononen J. Bubendorf L. Kallioniemi A. Barlund M. Schraml P. Leighton S. Torhorst J. Mihatsch M.J. Sauter G. Kallioniemi O.P. Tissue micro arrays for high-throughput molecular profiling of tumor specimens.Nat. Med. 1998; 4: 844-847Google Scholar, 3Hewitt S.M. Design, construction, and use of tissue micro arrays.Methods Mol. Biol. 2004; 264: 61-72Google Scholar). In addition, specific antibodies directed to a particular target protein allow numerous functional assays to be performed ranging from conventional ELISA assays to detailed localization studies using fluorescent probes and protein capture experiments (“pull-down”) for purification of specific proteins and their associated complexes for structural and biochemical analyses (4Agaton C. Uhlen M. Hober S. Genome-based proteomics.Electrophoresis. 2004; 25: 1280-1288Google Scholar, 5Tyers M. Mann M. From genomics to proteomics.Nature. 2003; 422: 193-197Google Scholar).The challenge for antibody-based proteomics is to move from a conventional protein-by-protein approach into a high throughput mode to allow chromosome wide analysis (6Hanash S. HUPO initiatives relevant to clinical proteomics.Mol. Cell. Proteomics. 2004; 3: 298-301Google Scholar, 7Celis J.E. Moreira J.M. Gromova I. Cabezon T. Ralfkiaer U. Guldberg P. Straten P.T. Mouridsen H. Friis E. Holm D. Rank F. Gromov P. Towards discovery-driven translational research in breast cancer.FEBS J. 2005; 272: 2-15Google Scholar). Technical challenges involve both the antigen production and the subsequent generation and characterization of the antibodies. In addition, methods for systematic protein profiling on a whole proteome level are lacking. Agaton et al. (8Agaton C. Galli J. Hoiden Guthenberg I. Janzon L. Hansson M. Asplund A. Brundell E. Lindberg S. Ruthberg I. Wester K. Wurtz D. Hoog C. Lundeberg J. Stahl S. Ponten F. Uhlen M. Affinity proteomics for systematic protein profiling of chromosome 21 gene products in human tissues.Mol. Cell. Proteomics. 2003; 2: 405-414Google Scholar) showed that the combination of the cloning and expression of recombinant protein fragments with immunohistochemistry analysis could be used for systematic protein expression and subcellular localization describing distribution and expression of putative gene products in normal tissues as well as in common cancers and other forms of diseased tissues. Recently Nilsson et al. (9Nilsson P. Paavilainen L. Larsson K. Ödling J. Sundberg M. Andersson A.-C. Kampf C. Persson A. Szigyarto C. A.-K. Ottosson J. Björling E. Hober S. Wernérus H. Wester K. Pontén F. Uhlen M. Towards a human proteome atlas: high-throughput generation of mono-specific antibodies for tissue profiling.Proteomics. 2005; (in press)Google Scholar) showed that this strategy could be further improved by a streamlined approach for affinity purification of the antibodies to generate monospecific antibodies (msAbs) 1The abbreviations used are: msAb, monospecific antibody; TMA, tissue microarray; PrEST, protein epitope signature tag; PBST, phosphate-buffered saline supplemented with Tween; TIFF, tagged image file format; JPEG, Joint Photographic Experts Group; HPR, human proteome resource; MAT1, menage a trois-1; CRIM-1, cysteine-rich motor neuron 1; CD31, cluster of differentiation 31; PSA, prostate-specific antigen. 1The abbreviations used are: msAb, monospecific antibody; TMA, tissue microarray; PrEST, protein epitope signature tag; PBST, phosphate-buffered saline supplemented with Tween; TIFF, tagged image file format; JPEG, Joint Photographic Experts Group; HPR, human proteome resource; MAT1, menage a trois-1; CRIM-1, cysteine-rich motor neuron 1; CD31, cluster of differentiation 31; PSA, prostate-specific antigen. and the subsequent validation of the specificity of these antibodies by protein microarrays.The use of tissue microarrays (TMAs) generated from multiple biopsies combined into single paraffin blocks enabled high throughput analysis of protein expression in various tissues and organs (2Kononen J. Bubendorf L. Kallioniemi A. Barlund M. Schraml P. Leighton S. Torhorst J. Mihatsch M.J. Sauter G. Kallioniemi O.P. Tissue micro arrays for high-throughput molecular profiling of tumor specimens.Nat. Med. 1998; 4: 844-847Google Scholar). Recently Kampf et al. (10Kampf C. Andersson A.C. Wester K. Bjorling E. Uhlen M. Ponten F. Antibody-based tissue profiling as a tool for clinical proteomics.Clin. Proteomics. 2004; 1: 285-299Google Scholar) showed that high throughput analysis of protein expression can be performed with a standard set of tissue microarrays representing both normal and cancer tissues. The TMA technology provides an automated array-based high throughput technique where as many as 1000 paraffin-embedded tissue samples can be brought into one paraffin block in an array format.We show that a comprehensive atlas of human protein expression patterns in normal and cancer tissues can be created by combining the methods mentioned above. A set of standardized TMAs was produced to allow for rapid screening of a multitude of different tissues and cell types using immunohistochemistry. Each antibody was used to screen a multitude of normal human tissues and cancer tissue from individually different tumors. Altogether 576 high resolution digital images corresponding to a total of 20 gigabytes of data were collected for each antibody.Both “in-house” generated monospecific antibodies and antibodies from commercial sources were used for the profiling of a large number of protein targets. Altogether more than 700 proteins were analyzed representing all major types of protein families, i.e. protein receptors, kinases, phosphatases, transcription factors, and nuclear receptors. The results are presented as a publicly available protein atlas database, and the data suggest that it should be possible to extend this analysis to most or all human proteins. This approach could also quite effectively be used for generation of expression data for model animals such as mouse, rat, and chimpanzee. A valuable tool for medical and biological research can thus be envisioned as a complement to genome and transcript profiling data.EXPERIMENTAL PROCEDURESGeneration of Antigens—Suitable protein epitope signature tags (PrESTs), representing unique regions for each target protein, were designed using bioinformatic tools (12Lindskog M. Rockberg J. Uhlen M. Sterky F. Selection of protein epitopes for antibody production.BioTechniques. 2005; 38: 723-727Google Scholar) and with the human genome sequence as template (EnsEMBL database). In the design of the PrESTs, transmembrane regions and signal peptides were avoided, and an amino acid sequence with a size between 100 and 150 amino acids and low homology to other human proteins was selected to decrease the risk of cross-reactivity of antibodies to other human proteins. The cloning, protein expression, immunization, and affinity purification to yield monospecific antibodies were performed as described previously by Agaton et al. (8Agaton C. Galli J. Hoiden Guthenberg I. Janzon L. Hansson M. Asplund A. Brundell E. Lindberg S. Ruthberg I. Wester K. Wurtz D. Hoog C. Lundeberg J. Stahl S. Ponten F. Uhlen M. Affinity proteomics for systematic protein profiling of chromosome 21 gene products in human tissues.Mol. Cell. Proteomics. 2003; 2: 405-414Google Scholar) and Nilsson et al. (9Nilsson P. Paavilainen L. Larsson K. Ödling J. Sundberg M. Andersson A.-C. Kampf C. Persson A. Szigyarto C. A.-K. Ottosson J. Björling E. Hober S. Wernérus H. Wester K. Pontén F. Uhlen M. Towards a human proteome atlas: high-throughput generation of mono-specific antibodies for tissue profiling.Proteomics. 2005; (in press)Google Scholar).PrEST Arrays—The PrESTs were diluted to 40 μg/ml in 0.1 m urea and 1× PBS (pH 7.4), and 50 μl of each PrEST was transferred to a 96-well spotting plate. The PrESTs were subsequently spotted and immobilized onto epoxide slides (Corning Life Sciences) using a pin-and-ring arrayer (Affymetrix 427). The slides were washed in 1× PBS (5 min), and then the surface was blocked with SuperBlock (Pierce) for 30 min. An adhesive 16-well silicone mask (Schleicher & Schuell) was applied to the glass before addition of the msAb diluted 1:2000 in 1× PBST (1× PBS, 0.1% Tween 20, pH 7.4) to a final concentration of approximately 50 ng/ml. Hen-generated tag-specific antibodies recovered from the depletion step were co-incubated with the monospecific antibodies, and the glass slides were incubated on a shaker for 60 min. The slides were washed with 1× PBST and 1× PBS two times for 10 min each before the secondary antibodies goat-anti rabbit Alexa 647 and goat anti-chicken Alexa 555 (Molecular Probes) diluted 1:60,000 to 30 ng/ml in 1× PBST were added and incubated for 60 min. After the same washing procedure as for the first incubation, the slides were spun dry and scanned using a G2565BA array scanner (Agilent), and images were quantified using the image analysis software GenePix 5.1 (Axon Instruments).Western Blots—Western blot analysis of affinity-purified antibodies was performed by separation of total protein extracts from selected human cell lines and tissues on precast 10–20% Criterion™ SDS-PAGE gradient gels (Bio-Rad) under reducing conditions followed by electrotransfer to PVDF membranes (Bio-Rad) according to the manufacturer’s recommendations. The membranes were blocked (5% dry milk, 0.5% Tween 20, 1× TBS, 0.1 m Tris-HCl, 0.5 m NaCl) for 1 h at room temperature, incubated with the primary antibody diluted 1:500 in blocking buffer, and washed in Tris-buffered saline supplemented with Tween 20. The secondary horseradish peroxidase-conjugated antibody (swine anti-rabbit immunoglobulin/horseradish peroxidase, Dakocytomation, Glostrup, Denmark) was diluted 1:3000 in blocking buffer, and chemiluminescence detection was carried out using a Chemidoc charge-couple device camera (Bio-Rad) and SuperSignal® West Dura Extended Duration substrate (Pierce) according to the manufacturer’s protocol.Immunohistochemistry—Slides were baked for 45 min in 60 °C, deparaffinized in xylene, hydrated in graded alcohols, and blocked for endogenous peroxidase in 0.3% hydrogen peroxide diluted in 80% ethanol. For antigen retrieval, a Decloaking chamber (Biocare Medical, Walnut Creek, CA) was used. Slides were immersed and boiled in Target Retrieval Solution, pH 6.0 (Dakocytomation) for 4 min at 125 °C and then allowed to cool down to 90 °C. Automated immunohistochemistry was done using an Autostainer Plus instrument (Dakocytomation). Primary antibodies and a dextran polymer visualization system (Envision, Dakocytomation) were incubated for 30 min each at room temperature, and slides were developed for 2 × 5 min using diaminobenzidine (Dakocytomation) as chromogen. Each incubation was followed by rinsing in wash buffer (Dakocytomation). After a short rinse in tap water, slides were counterstained in Harris hematoxylin (Sigma) and coverslipped using Pertex (Histolab, Gothenburg, Sweden) as mounting medium.Digital Imaging of the Tissue Cores—All immunohistochemically stained sections from the eight different TMAs were scanned using an automated slide-scanning system, ScanScope T2 (Aperio Technologies, Vista, CA). For each antibody 576 digital images were generated to represent the total content of the eight TMAs. Scanning was performed at 20× magnification. Digital images were separated and extracted as individual TIFF files for storage of original data. The size of each TIFF image is ∼20–30 megabytes. The high resolution TIFF images are stored on digital tapes, and to facilitate handling the images in a web-based annotation system the individual, images were compressed from TIFF format into JPEG format.Scoring of Protein Expression—The annotation software was developed to allow for a basic and rapid evaluation of immunoreactivity in a broad spectrum of different tissues and cell types. The annotation tool software was developed to run on any standard desktop personal computer (Microsoft Windows and Macintosh operating systems) using an ordinary web browser interface. Parameters that were annotated included overall staining, congruity in staining between triplicate/duplicate samples, validation of immunohistochemical staining as well as staining intensity, fraction of immunoreactive cells, and pattern and localization of immunoreactivity (nuclear, cytoplasmic, or cell membranous). A text box was also included for comments.Information Technology—The protein atlas is a web-based service clustered on multiple web servers, each serving the web pages, database, and all the images individually due to “fail-safe” demands. All software used within the protein atlas are open source based on the LAMP setup (Linux, Apache, MySQL, and PHP). The protein atlas is loaded with data and image files from the HPR-LIMS (Laboratory Information Management System), which is the production system especially developed for and used within the HPR project.RESULTSThe Monospecific Antibodies Used in the Protein Atlas—In this study, we generated monospecific antibodies by a strategy where PrESTs are used both as antigens for the development of polyclonal antibodies and as affinity ligands for the subsequent affinity purification of the antibodies as described before (8Agaton C. Galli J. Hoiden Guthenberg I. Janzon L. Hansson M. Asplund A. Brundell E. Lindberg S. Ruthberg I. Wester K. Wurtz D. Hoog C. Lundeberg J. Stahl S. Ponten F. Uhlen M. Affinity proteomics for systematic protein profiling of chromosome 21 gene products in human tissues.Mol. Cell. Proteomics. 2003; 2: 405-414Google Scholar, 9Nilsson P. Paavilainen L. Larsson K. Ödling J. Sundberg M. Andersson A.-C. Kampf C. Persson A. Szigyarto C. A.-K. Ottosson J. Björling E. Hober S. Wernérus H. Wester K. Pontén F. Uhlen M. Towards a human proteome atlas: high-throughput generation of mono-specific antibodies for tissue profiling.Proteomics. 2005; (in press)Google Scholar, 11Agaton C. Falk R. Hoiden Guthenberg I. Gostring L. Uhlen M. Hober S. Selective enrichment of monospecific polyclonal antibodies for antibody-based proteomics efforts.J. Chromatogr. A. 2004; 1043: 33-40Google Scholar, 12Lindskog M. Rockberg J. Uhlen M. Sterky F. Selection of protein epitopes for antibody production.BioTechniques. 2005; 38: 723-727Google Scholar). In addition, we used commercially available antibodies selected primarily by their medical relevance and importance. Altogether 718 antibodies representing all human chromosomes were used to create the protein atlas, although the focus was on chromosomes 14, 22, and X for the monospecific antibodies (Fig. 1).Validation of the Antibodies—The monospecific antibodies were validated using a broad range of quality assurance analyses. All cloned gene fragments were sequence-verified, and the recombinant produced antigens (PrESTs) were analyzed by electrospray mass spectrometry to verify the expected molecular weight (data not shown). The affinity-purified antibodies were analyzed by a novel protein array method (9Nilsson P. Paavilainen L. Larsson K. Ödling J. Sundberg M. Andersson A.-C. Kampf C. Persson A. Szigyarto C. A.-K. Ottosson J. Björling E. Hober S. Wernérus H. Wester K. Pontén F. Uhlen M. Towards a human proteome atlas: high-throughput generation of mono-specific antibodies for tissue profiling.Proteomics. 2005; (in press)Google Scholar) in which a multitude of human protein fragments (PrESTs) were spotted on a single glass slide. This assay, using fluorescently labeled secondary antibodies for signal detection, provides information about the specificity and purity of the monospecific antibodies. In Fig. 2, four examples of this analysis are shown in which 1440 PrEST fragments have been spotted in triplicates on glass slides, and the signal of the antibody binding to each spot is shown. Furthermore the figure shows two separate examples of the results from the protein array analysis for duplicate antibody fractions obtained by immunizing two separate animals with the same antigen (Fig. 2, a/b and c/d, respectively). The results show that the antibodies are in each case specific for the antigen and also that reproducible antibodies, as judged by the protein array, can be obtained by immunization with the same antigen in two separate animals.Fig. 2Protein microarrays for validation of antibodies. The results of the specificity analysis of two pairs of monospecific antibodies on protein arrays with 1440 PrESTs are shown. Each pair (a and b and c and d) corresponds to antibodies generated against the same antigen in two different animals. The red bar corresponds to the relative signal from the specific antigen, and the black bars represent the cross-reactivity to the other PrESTs. In each graph is an image from one-fourth of the array inserted. A dual color system is used where the presence of all proteins is detected in green, and the specific antibody is detected in red. a and b, antibodies HPR000534 and HPR001102 directed against a DnaJ homolog protein. c and d, antibodies HPR000830 and HPR001088 directed against a predicted KIAA protein.View Large Image Figure ViewerDownload (PPT)In most cases, the monospecific antibodies were further validated by Western blotting with total protein extracts from selected human cell lines and tissues. These analyses give important data about protein size and expression patterns. The standard design of the Western blots contains three human cell lines (RT-4, EFO-21, and A-431) and two human tissues (liver and tonsil). In many cases, the analysis showed a specific staining with a single band corresponding to the size of the expected human protein as exemplified by four antibodies toward four different proteins with an expected molecular mass of 61, 22, 126, and 57 kDa, respectively (Fig. 3, a–d). For other antibodies additional bands were detected (Fig. 3e), and in some cases no band of the correct size could be seen (Fig. 3f). It should be noted that the lack of the expected protein or the presence of additional bands could be explained by the expression pattern for the investigated protein or by protein modifications, proteolysis, or the presence of unknown splice variants.Fig. 3Tissue Western analysis of purified monospecific antibodies. Total protein Western blots of a number of antibodies using protein extracts from human cell lines (RT-4, EFO-21, and A-431) and tissues (liver and tonsil) are shown. The filled arrow indicates the size of the expected protein as determined from the genome sequence. a, antibody HPR000427 generated toward the 61-kDa cleavage stimulation factor subunit encoded by the CSTF2 gene located on the X chromosome. b, antibody HPR000722 raised against the 22-kDa BH3-interacting domain death agonist (BID) transcribed from chromosome 22. c, HPR001122 recognizing the 126-kDa WD repeat and high mobility group box DNA-binding protein 1 encoded by the WDHD1gene on chromosome 14. d, HPR000527 binding to an unknown 57-kDa protein. e, HPR000798 generated toward the heat shock-related 70-kDa protein 2 gene HSPA2 located on chromosome 14. f, HPR00129 designed toward the snRNA-activating protein complex 43-kDa subunit. MWM, molecular weight marker.View Large Image Figure ViewerDownload (PPT)All antibodies were further used to stain a tissue microarray with human tissues and organs. If possible, the immunohistochemistry pattern was compared with bioinformatic data based on gene, transcript profiling, protein expression, and localization data. In addition, for selected antibodies, a competition-based adsorption assay was carried out as described previously (9Nilsson P. Paavilainen L. Larsson K. Ödling J. Sundberg M. Andersson A.-C. Kampf C. Persson A. Szigyarto C. A.-K. Ottosson J. Björling E. Hober S. Wernérus H. Wester K. Pontén F. Uhlen M. Towards a human proteome atlas: high-throughput generation of mono-specific antibodies for tissue profiling.Proteomics. 2005; (in press)Google Scholar). The assay was performed by incubation of the antibody with its antigen before immunostaining. Disappearance of the specific staining was interpreted as evidence that the observed immunohistochemistry pattern was specific and that the msAb recognized the expected target.Evaluation of the complete set of validation assays showed that approximately half of the antibodies exhibited possible cross-reactivity and were consequently excluded from the protein atlas database. A validation score was given for each of the remaining monospecific antibodies. A high validation score indicates that the quality assurance supports, with high confidence, the specificity of the msAb toward the expected human target protein. A low validation score shows that the antibody is probably specific to the expected target, but in this case, the validation is less clear, and cross-reactivity cannot be excluded. For the 275 msAbs analyzed in this study, 160 have a low validation score, whereas 68 and 47 have a medium or high validation score, respectively. For the 443 commercial antibodies, the only validation performed as part of the project was a comparison between our data and the data obtained from the antibody provider and a bioinformatic comparison between our experimental data from the tissue microarrays with expected tissue profiles as judged from the literature.Immunohistochemistry and Image Analysis—Once the high throughput antibody-based tissue profiling was available, it became feasible to create an atlas of protein expression patterns in a multitude of normal human tissues and cancer tissues representing the 20 most prevalent cancer types. A set of standardized TMAs was produced as described by Kampf et al. (10Kampf C. Andersson A.C. Wester K. Bjorling E. Uhlen M. Ponten F. Antibody-based tissue profiling as a tool for clinical proteomics.Clin. Proteomics. 2004; 1: 285-299Google Scholar) containing 48 different human tissues in triplicate and cancer tissues from 216 individually different tumors in duplicate. Digital images for annotation of expression profiles were generated using a semiautomated approach (10Kampf C. Andersson A.C. Wester K. Bjorling E. Uhlen M. Ponten F. Antibody-based tissue profiling as a tool for clinical proteomics.Clin. Proteomics. 2004; 1: 285-299Google Scholar, 13Vrolijk H. Sloos W. Mesker W. Franken P. Fodde R. Morreau H. Tanke H. Automated acquisition of stained tissue micro arrays for high-throughput evaluation of molecular targets.J. Mol. Diagn. 2003; 5: 160-167Google Scholar), and 576 images corresponding to a total of 20 gigabytes of data were collected for each antibody. The original TIFF images were stored on digital tapes for future analysis, and the images were converted to JPEG images suitable for web-based browsing. Each JPEG image corresponds to ∼1 megabyte of data with the image compression ratio adequate for analysis down to subcellular levels.The images were annotated by certified pathologists using a newly designed web-based annotation software. 2E. Björling and P. Oksvold, unpublished data. The manual annotation of each image provides a knowledge base for functional studies and allows queries about protein profiles in normal and disease tissues. Parameters that were annotated included overall staining, congruity in staining between triplicate/duplicate samples, and validation of immunohistochemistry staining. Staining intensity, fraction of immunoactive cells, and patterns and localization of immunoreactivity, such as nuclear, cytoplasm, or membranous, were also noted (10Kampf C. Andersson A.C. Wester K. Bjorling E. Uhlen M. Ponten F. Antibody-based tissue profiling as a tool for clinical proteomics.Clin. Proteomics. 2004; 1: 285-299Google Scholar).The initial annotation was performed during a 2-day “annotation jamboree” in which 26 pathologists from Sweden, Norway, and Finland gathered and annotated ∼80,000 images using individual desktop terminals. Some antibodies were annotated independently by two or more pathologists. This allowed analysis of consistency and comparability of annotations from different pathologists. The workshop was subsequently followed by an internet-based system in which each pathologist could book a particular antibody, download the corresponding 576 images, perform the annotation, and then submit back the annotation to the central database. In this way, all the 400,000 images in the protein atlas were annotated.Protein Atlas for Normal Tissues—The publicly available database (www.proteinatlas.org) contains, in the first version, more than 400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. The protein profiles in normal tissues of a particular protein are displayed by a summary page including all 48 tissues analyzed (Fig. 4, a and b). Intensity and abundance of immunoreactivity are given as a color code from white (no protein presence) to red (high amounts of protein). Each colored circle represents an annotated tissue type, and the circles can be clicked to show the underlying original images.Fig. 4The summary page for normal protein profiles. Two examples of the summary page for normal tissue profiles in 48 different normal tissues are shown. Intensity and abundance of immunoreactivity is given as a color code (red = strong, orange = moderate, yellow = weak, white = no staining, and black = missing tissue). Each colored circle represents one tissue type. The circles are divided in three because each tissue is represented by samples from three different patients. Two examples of normal tissue profiles obtained with antibodies are shown: a, the antibody HPR000701 directed toward the MAT1 gene product from chromosome 14; and b, the antibody HPR001012 directed toward the ARHGAP4gene product from the X chromosome. One example of a TMA spot from normal testis stained with an antibody generated from the MAT1gene is shown at low (c) and at high magnification (d). Similarly a TMA spot showing immunohistochemical outcome from the ARHGAP4 gene is shown at low (e) and high magnification (f).View Large Image Figure ViewerDownload (PPT)The summary results from the antibody HPR000701 toward the cyclin-dependent kinase-activating kinase assembly factor MAT1 protein show a weak or moderate expression pattern in a majority of analyzed cell types, whereas a strong expression is detected in testis and urinary bladder (Fig. 4a). This gene product contains a type 1 RING-type zinc finger, and the protein is reported to be involved in cell cycle control and RNA transcription by RNA polymerase II (14Tirode F. Busso D. Coin F. Egly J.M. Reconstitution of the transcription factor TFIIH: assignment of functions for the three enzymatic subunits, XPB, XPD, and cdk7.Mol. Cell. 1999; 3: 87-95Google Scholar, 15Gervais V. Busso D. Wasielewski E. Poterszman A. Egly J.M. Thierry J.C. Kieffer B. Solution structure of the N-terminal domain of the human TFIIH MAT1 subunit: new insights into the RING finger family.J. Biol. Chem. 2001; 276: 7457-7464Google Scholar). Highest levels of expression have been reported in colon and testis (16Perez C. Auriol J. Seroz T. Egly J.M. Genomic organization and promoter characterization of the mouse an human genes encoding p62 subunit of the transcription/DNA repair factor TFIIH.Gene (Amst.). 1998; 213: 73-82Google Scholar) supporting the protein expression data. The underlying image for testis (Fig. 4c) with its “microscope” view (Fig. 4d) shows strong nuclear staining in germ cells in the seminiferous duct.In Fig.
Год издания: 2005
Авторы: Mathias Uhlén, Erik Björling, Charlotta Agaton, Cristina Al‐Khalili Szigyarto, Bahram Amini, Elisabet Andersen, Ann‐Catrin Andersson, Pia Angelidou, Anna Asplund, Caroline Asplund, Lisa Berglund, Kristina Bergström, Harry Brumer, Dijana Cerjan, Marica Ekström, Adila El‐Obeid, Cecilia Eriksson, Linn Fagerberg, Ronny Falk, Jenny Fall, Mattias Forsberg, Marcus Björklund, Kristoffer Gumbel, Asif Halimi, Inga Hallin, Carl Hamsten, Marianne Hansson, My Hedhammar, Görel Hercules, Caroline Kampf, Karin Larsson, Mats Lindskog, Wald Lodewyckx, J. Lund, Joakim Lundeberg, Kristina Magnusson, Erik Malm, Peter Nilsson, Jenny Ödling, Per Oksvold, Ing‐Marie Olsson, Emma Öster, Jenny Ottosson, Linda Paavilainen, Anja Persson, Rebecca Rimini, Johan Rockberg, Marcus Runeson, Åsa Sivertsson, Anna Sköllermo, Johanna Stéen, Maria Stenvall, Fredrik Sterky, Sara Strömberg, Mårten Sundberg, Hanna Tegel, Samuel Tourle, Eva Wahlund, Annelie Waldén, Jinghong Wan, Henrik Wernérus, Joakim Westberg, Kenneth Wester, Ulla Wrethagen, Lan Xu, Sophia Hober, Fredrik Pontén
Издательство: Elsevier BV
Источник: Molecular & Cellular Proteomics
Ключевые слова: Monoclonal and Polyclonal Antibodies Research, Advanced Proteomics Techniques and Applications, Advanced Biosensing Techniques and Applications
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Том: 4
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Страницы: 1920–1932