Expression, localization and clinical application of exogenous Smith proteins D1 in gene transfected HEp-2 cells
Aim To establish an improved substrate for an indirect immunofluorescence test (IIF) to detect anti‐Sm antibody. Methods Full‐length Smith protein D1(Sm‐D1) complementary DNA was obtained from human larynx carcinoma cell line HEp‐2 by reverse transcription – polymerase chain reaction (RT‐PCR) and cl...
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| Veröffentlicht in: | International journal of rheumatic diseases 2013-06, Vol.16 (3), p.303-309 |
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| creator | Wang, Su-li Wang, Fang-fang Chen, Shun-le Shen, Nan Xue, Feng Ye, Ping Bao, Chun-de Gu, Yue-ying Yu, Chong-zhao Wilson, Alisa Wallace, Daniel J. Weisman, Michael H. Lu, Liang-jing |
| description | Aim
To establish an improved substrate for an indirect immunofluorescence test (IIF) to detect anti‐Sm antibody.
Methods
Full‐length Smith protein D1(Sm‐D1) complementary DNA was obtained from human larynx carcinoma cell line HEp‐2 by reverse transcription – polymerase chain reaction (RT‐PCR) and cloned into the mammalian expression vector pEGFP‐C1. The recombinant plasmid pEGFP‐Sm‐D1 was transfected into HEp‐2 cells. The expression, localization and antigenicity of fusion proteins of green fluorescent protein (GFP) in transfected cells were confirmed by means of immunoblotting (IBT), confocal fluorescence microscopy and IIF analysis. Transfected HEp‐2 cells were analyzed with reference serum and compared with untransfected HEp‐2 cells by IIF.
Results
Stable expression of the Sm‐D1‐GFP was maintained for more than ten generations. This Sm‐D1‐GFP showed the antigenicity of Sm‐D1 with a characteristic phenotype in IIF.Six of 12 serum specimens from systemic lupus erythematosus contained both 29/28 and 13.5 kDa proteins and showed characteristic immunofluorescent patterns. The same phenomenon appeared in 3/6 serum samples which contained 29/28 kDa proteins only. Sera from 10 healthy donors did not react with HEp‐Sm‐D1 or HEp‐2 at 1:80 attenuant degrees. No alteration in expression, localization and morphology was observed when HEp‐Sm‐D1 or HEp‐2 interacted with the reference sera which could react with Ro/SSA, La/SSB, β2GP1, centromere, histone, and Scl‐70 antibodies in routine IIF tests.
Conclusion
As a new kind of substrate of IIF, HEp‐Sm‐D1 can be used to detect anti‐Sm antibodies. Transfected HEp‐2 cells keep the immunofluorescent property of HEp‐2 cells in immunofluorescence anti‐nuclear antibody (IFANA) test and could potentially be used as substrate for routine IFANA detection. |
| doi_str_mv | 10.1111/1756-185X.12000 |
| format | Article |
| fullrecord | <record><control><sourceid>proquest_pubme</sourceid><recordid>TN_cdi_proquest_miscellaneous_1855084392</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>1855084392</sourcerecordid><originalsourceid>FETCH-LOGICAL-i4260-e0fc3ab98f897b358de6334bd3c3ad2a7ccd08857047a86e54d1a69661afdf33</originalsourceid><addsrcrecordid>eNqFUU1PGzEUtBBVoWnP3JAlLhxYaq8_94hCSFpFLRKRys1y1m-p6ca7rDci9NfXm9AcuOCLx2_m2eM3CJ1QcknT-kqVkBnV4v6S5oSQA3S8rxzuMadH6FOMj4RIyqT6iI5yVuhE5seonWzaDmL0TbjAdVPa2v-1fTphGxwuax98qmHbtnUCW6KpMGyaBwjNOuK7le9_47ZrevAh4muKfcCJA9x3NsQKyh4cnk3aLMcl1HX8jD5Uto7w5XUfocXNZDGeZfOf02_jq3nmeS5JBqQqmV0WutKFWjKhHUjG-NKxVHa5VWXpiNZCEa6sliC4o1YWUlJbuYqxETrfXZusPa0h9mbl42DABki-TZqQIJqzIn9fynOtFGVaJ-nZG-ljs-5C-oehgvNCFIwPb5--qtbLFTjTdn5luxfzf-pJIHaCZ1_Dy56nxAyhmiG2weC92YZqrm7nW5D6sl2fjz1s9n22-2OkYkqYXz-mht7OF3ffr6WZsX9eRqIm</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>1544959343</pqid></control><display><type>article</type><title>Expression, localization and clinical application of exogenous Smith proteins D1 in gene transfected HEp-2 cells</title><source>MEDLINE</source><source>Access via Wiley Online Library</source><creator>Wang, Su-li ; Wang, Fang-fang ; Chen, Shun-le ; Shen, Nan ; Xue, Feng ; Ye, Ping ; Bao, Chun-de ; Gu, Yue-ying ; Yu, Chong-zhao ; Wilson, Alisa ; Wallace, Daniel J. ; Weisman, Michael H. ; Lu, Liang-jing</creator><creatorcontrib>Wang, Su-li ; Wang, Fang-fang ; Chen, Shun-le ; Shen, Nan ; Xue, Feng ; Ye, Ping ; Bao, Chun-de ; Gu, Yue-ying ; Yu, Chong-zhao ; Wilson, Alisa ; Wallace, Daniel J. ; Weisman, Michael H. ; Lu, Liang-jing</creatorcontrib><description>Aim
To establish an improved substrate for an indirect immunofluorescence test (IIF) to detect anti‐Sm antibody.
Methods
Full‐length Smith protein D1(Sm‐D1) complementary DNA was obtained from human larynx carcinoma cell line HEp‐2 by reverse transcription – polymerase chain reaction (RT‐PCR) and cloned into the mammalian expression vector pEGFP‐C1. The recombinant plasmid pEGFP‐Sm‐D1 was transfected into HEp‐2 cells. The expression, localization and antigenicity of fusion proteins of green fluorescent protein (GFP) in transfected cells were confirmed by means of immunoblotting (IBT), confocal fluorescence microscopy and IIF analysis. Transfected HEp‐2 cells were analyzed with reference serum and compared with untransfected HEp‐2 cells by IIF.
Results
Stable expression of the Sm‐D1‐GFP was maintained for more than ten generations. This Sm‐D1‐GFP showed the antigenicity of Sm‐D1 with a characteristic phenotype in IIF.Six of 12 serum specimens from systemic lupus erythematosus contained both 29/28 and 13.5 kDa proteins and showed characteristic immunofluorescent patterns. The same phenomenon appeared in 3/6 serum samples which contained 29/28 kDa proteins only. Sera from 10 healthy donors did not react with HEp‐Sm‐D1 or HEp‐2 at 1:80 attenuant degrees. No alteration in expression, localization and morphology was observed when HEp‐Sm‐D1 or HEp‐2 interacted with the reference sera which could react with Ro/SSA, La/SSB, β2GP1, centromere, histone, and Scl‐70 antibodies in routine IIF tests.
Conclusion
As a new kind of substrate of IIF, HEp‐Sm‐D1 can be used to detect anti‐Sm antibodies. Transfected HEp‐2 cells keep the immunofluorescent property of HEp‐2 cells in immunofluorescence anti‐nuclear antibody (IFANA) test and could potentially be used as substrate for routine IFANA detection.</description><identifier>ISSN: 1756-1841</identifier><identifier>EISSN: 1756-185X</identifier><identifier>DOI: 10.1111/1756-185X.12000</identifier><identifier>PMID: 23981752</identifier><language>eng</language><publisher>England: Blackwell Publishing Ltd</publisher><subject>Antigen-Antibody Reactions ; antigenic localization ; Autoantibodies - blood ; Biomarkers - blood ; Blotting, Western ; Carcinoma - genetics ; Carcinoma - metabolism ; Case-Control Studies ; Cell Line, Tumor ; Cloning, Molecular ; Epitopes ; Green Fluorescent Proteins - genetics ; Green Fluorescent Proteins - metabolism ; HEp-2 cells ; Humans ; indirect immunofluorescent technique ; Laryngeal Neoplasms - genetics ; Laryngeal Neoplasms - metabolism ; Lupus Erythematosus, Systemic - blood ; Lupus Erythematosus, Systemic - diagnosis ; Lupus Erythematosus, Systemic - immunology ; Microscopy, Confocal ; Microscopy, Fluorescence ; Recombinant Fusion Proteins - genetics ; Recombinant Fusion Proteins - metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Sm-D1 ; snRNP Core Proteins - genetics ; snRNP Core Proteins - metabolism ; Transfection</subject><ispartof>International journal of rheumatic diseases, 2013-06, Vol.16 (3), p.303-309</ispartof><rights>2012 The Authors International Journal of Rheumatic Diseases © 2012 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd</rights><rights>2012 The Authors International Journal of Rheumatic Diseases © 2012 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd.</rights><rights>International Journal of Rheumatic Diseases © 2012 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1111%2F1756-185X.12000$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1111%2F1756-185X.12000$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>315,781,785,1418,27928,27929,45578,45579</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/23981752$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Wang, Su-li</creatorcontrib><creatorcontrib>Wang, Fang-fang</creatorcontrib><creatorcontrib>Chen, Shun-le</creatorcontrib><creatorcontrib>Shen, Nan</creatorcontrib><creatorcontrib>Xue, Feng</creatorcontrib><creatorcontrib>Ye, Ping</creatorcontrib><creatorcontrib>Bao, Chun-de</creatorcontrib><creatorcontrib>Gu, Yue-ying</creatorcontrib><creatorcontrib>Yu, Chong-zhao</creatorcontrib><creatorcontrib>Wilson, Alisa</creatorcontrib><creatorcontrib>Wallace, Daniel J.</creatorcontrib><creatorcontrib>Weisman, Michael H.</creatorcontrib><creatorcontrib>Lu, Liang-jing</creatorcontrib><title>Expression, localization and clinical application of exogenous Smith proteins D1 in gene transfected HEp-2 cells</title><title>International journal of rheumatic diseases</title><addtitle>Int J Rheum Dis</addtitle><description>Aim
To establish an improved substrate for an indirect immunofluorescence test (IIF) to detect anti‐Sm antibody.
Methods
Full‐length Smith protein D1(Sm‐D1) complementary DNA was obtained from human larynx carcinoma cell line HEp‐2 by reverse transcription – polymerase chain reaction (RT‐PCR) and cloned into the mammalian expression vector pEGFP‐C1. The recombinant plasmid pEGFP‐Sm‐D1 was transfected into HEp‐2 cells. The expression, localization and antigenicity of fusion proteins of green fluorescent protein (GFP) in transfected cells were confirmed by means of immunoblotting (IBT), confocal fluorescence microscopy and IIF analysis. Transfected HEp‐2 cells were analyzed with reference serum and compared with untransfected HEp‐2 cells by IIF.
Results
Stable expression of the Sm‐D1‐GFP was maintained for more than ten generations. This Sm‐D1‐GFP showed the antigenicity of Sm‐D1 with a characteristic phenotype in IIF.Six of 12 serum specimens from systemic lupus erythematosus contained both 29/28 and 13.5 kDa proteins and showed characteristic immunofluorescent patterns. The same phenomenon appeared in 3/6 serum samples which contained 29/28 kDa proteins only. Sera from 10 healthy donors did not react with HEp‐Sm‐D1 or HEp‐2 at 1:80 attenuant degrees. No alteration in expression, localization and morphology was observed when HEp‐Sm‐D1 or HEp‐2 interacted with the reference sera which could react with Ro/SSA, La/SSB, β2GP1, centromere, histone, and Scl‐70 antibodies in routine IIF tests.
Conclusion
As a new kind of substrate of IIF, HEp‐Sm‐D1 can be used to detect anti‐Sm antibodies. Transfected HEp‐2 cells keep the immunofluorescent property of HEp‐2 cells in immunofluorescence anti‐nuclear antibody (IFANA) test and could potentially be used as substrate for routine IFANA detection.</description><subject>Antigen-Antibody Reactions</subject><subject>antigenic localization</subject><subject>Autoantibodies - blood</subject><subject>Biomarkers - blood</subject><subject>Blotting, Western</subject><subject>Carcinoma - genetics</subject><subject>Carcinoma - metabolism</subject><subject>Case-Control Studies</subject><subject>Cell Line, Tumor</subject><subject>Cloning, Molecular</subject><subject>Epitopes</subject><subject>Green Fluorescent Proteins - genetics</subject><subject>Green Fluorescent Proteins - metabolism</subject><subject>HEp-2 cells</subject><subject>Humans</subject><subject>indirect immunofluorescent technique</subject><subject>Laryngeal Neoplasms - genetics</subject><subject>Laryngeal Neoplasms - metabolism</subject><subject>Lupus Erythematosus, Systemic - blood</subject><subject>Lupus Erythematosus, Systemic - diagnosis</subject><subject>Lupus Erythematosus, Systemic - immunology</subject><subject>Microscopy, Confocal</subject><subject>Microscopy, Fluorescence</subject><subject>Recombinant Fusion Proteins - genetics</subject><subject>Recombinant Fusion Proteins - metabolism</subject><subject>Reverse Transcriptase Polymerase Chain Reaction</subject><subject>Sm-D1</subject><subject>snRNP Core Proteins - genetics</subject><subject>snRNP Core Proteins - metabolism</subject><subject>Transfection</subject><issn>1756-1841</issn><issn>1756-185X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2013</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFUU1PGzEUtBBVoWnP3JAlLhxYaq8_94hCSFpFLRKRys1y1m-p6ca7rDci9NfXm9AcuOCLx2_m2eM3CJ1QcknT-kqVkBnV4v6S5oSQA3S8rxzuMadH6FOMj4RIyqT6iI5yVuhE5seonWzaDmL0TbjAdVPa2v-1fTphGxwuax98qmHbtnUCW6KpMGyaBwjNOuK7le9_47ZrevAh4muKfcCJA9x3NsQKyh4cnk3aLMcl1HX8jD5Uto7w5XUfocXNZDGeZfOf02_jq3nmeS5JBqQqmV0WutKFWjKhHUjG-NKxVHa5VWXpiNZCEa6sliC4o1YWUlJbuYqxETrfXZusPa0h9mbl42DABki-TZqQIJqzIn9fynOtFGVaJ-nZG-ljs-5C-oehgvNCFIwPb5--qtbLFTjTdn5luxfzf-pJIHaCZ1_Dy56nxAyhmiG2weC92YZqrm7nW5D6sl2fjz1s9n22-2OkYkqYXz-mht7OF3ffr6WZsX9eRqIm</recordid><startdate>201306</startdate><enddate>201306</enddate><creator>Wang, Su-li</creator><creator>Wang, Fang-fang</creator><creator>Chen, Shun-le</creator><creator>Shen, Nan</creator><creator>Xue, Feng</creator><creator>Ye, Ping</creator><creator>Bao, Chun-de</creator><creator>Gu, Yue-ying</creator><creator>Yu, Chong-zhao</creator><creator>Wilson, Alisa</creator><creator>Wallace, Daniel J.</creator><creator>Weisman, Michael H.</creator><creator>Lu, Liang-jing</creator><general>Blackwell Publishing Ltd</general><general>Wiley Subscription Services, Inc</general><scope>BSCLL</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>7QP</scope><scope>7T5</scope><scope>H94</scope><scope>7X8</scope></search><sort><creationdate>201306</creationdate><title>Expression, localization and clinical application of exogenous Smith proteins D1 in gene transfected HEp-2 cells</title><author>Wang, Su-li ; Wang, Fang-fang ; Chen, Shun-le ; Shen, Nan ; Xue, Feng ; Ye, Ping ; Bao, Chun-de ; Gu, Yue-ying ; Yu, Chong-zhao ; Wilson, Alisa ; Wallace, Daniel J. ; Weisman, Michael H. ; Lu, Liang-jing</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-i4260-e0fc3ab98f897b358de6334bd3c3ad2a7ccd08857047a86e54d1a69661afdf33</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2013</creationdate><topic>Antigen-Antibody Reactions</topic><topic>antigenic localization</topic><topic>Autoantibodies - blood</topic><topic>Biomarkers - blood</topic><topic>Blotting, Western</topic><topic>Carcinoma - genetics</topic><topic>Carcinoma - metabolism</topic><topic>Case-Control Studies</topic><topic>Cell Line, Tumor</topic><topic>Cloning, Molecular</topic><topic>Epitopes</topic><topic>Green Fluorescent Proteins - genetics</topic><topic>Green Fluorescent Proteins - metabolism</topic><topic>HEp-2 cells</topic><topic>Humans</topic><topic>indirect immunofluorescent technique</topic><topic>Laryngeal Neoplasms - genetics</topic><topic>Laryngeal Neoplasms - metabolism</topic><topic>Lupus Erythematosus, Systemic - blood</topic><topic>Lupus Erythematosus, Systemic - diagnosis</topic><topic>Lupus Erythematosus, Systemic - immunology</topic><topic>Microscopy, Confocal</topic><topic>Microscopy, Fluorescence</topic><topic>Recombinant Fusion Proteins - genetics</topic><topic>Recombinant Fusion Proteins - metabolism</topic><topic>Reverse Transcriptase Polymerase Chain Reaction</topic><topic>Sm-D1</topic><topic>snRNP Core Proteins - genetics</topic><topic>snRNP Core Proteins - metabolism</topic><topic>Transfection</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Wang, Su-li</creatorcontrib><creatorcontrib>Wang, Fang-fang</creatorcontrib><creatorcontrib>Chen, Shun-le</creatorcontrib><creatorcontrib>Shen, Nan</creatorcontrib><creatorcontrib>Xue, Feng</creatorcontrib><creatorcontrib>Ye, Ping</creatorcontrib><creatorcontrib>Bao, Chun-de</creatorcontrib><creatorcontrib>Gu, Yue-ying</creatorcontrib><creatorcontrib>Yu, Chong-zhao</creatorcontrib><creatorcontrib>Wilson, Alisa</creatorcontrib><creatorcontrib>Wallace, Daniel J.</creatorcontrib><creatorcontrib>Weisman, Michael H.</creatorcontrib><creatorcontrib>Lu, Liang-jing</creatorcontrib><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Immunology Abstracts</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>International journal of rheumatic diseases</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Wang, Su-li</au><au>Wang, Fang-fang</au><au>Chen, Shun-le</au><au>Shen, Nan</au><au>Xue, Feng</au><au>Ye, Ping</au><au>Bao, Chun-de</au><au>Gu, Yue-ying</au><au>Yu, Chong-zhao</au><au>Wilson, Alisa</au><au>Wallace, Daniel J.</au><au>Weisman, Michael H.</au><au>Lu, Liang-jing</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Expression, localization and clinical application of exogenous Smith proteins D1 in gene transfected HEp-2 cells</atitle><jtitle>International journal of rheumatic diseases</jtitle><addtitle>Int J Rheum Dis</addtitle><date>2013-06</date><risdate>2013</risdate><volume>16</volume><issue>3</issue><spage>303</spage><epage>309</epage><pages>303-309</pages><issn>1756-1841</issn><eissn>1756-185X</eissn><abstract>Aim
To establish an improved substrate for an indirect immunofluorescence test (IIF) to detect anti‐Sm antibody.
Methods
Full‐length Smith protein D1(Sm‐D1) complementary DNA was obtained from human larynx carcinoma cell line HEp‐2 by reverse transcription – polymerase chain reaction (RT‐PCR) and cloned into the mammalian expression vector pEGFP‐C1. The recombinant plasmid pEGFP‐Sm‐D1 was transfected into HEp‐2 cells. The expression, localization and antigenicity of fusion proteins of green fluorescent protein (GFP) in transfected cells were confirmed by means of immunoblotting (IBT), confocal fluorescence microscopy and IIF analysis. Transfected HEp‐2 cells were analyzed with reference serum and compared with untransfected HEp‐2 cells by IIF.
Results
Stable expression of the Sm‐D1‐GFP was maintained for more than ten generations. This Sm‐D1‐GFP showed the antigenicity of Sm‐D1 with a characteristic phenotype in IIF.Six of 12 serum specimens from systemic lupus erythematosus contained both 29/28 and 13.5 kDa proteins and showed characteristic immunofluorescent patterns. The same phenomenon appeared in 3/6 serum samples which contained 29/28 kDa proteins only. Sera from 10 healthy donors did not react with HEp‐Sm‐D1 or HEp‐2 at 1:80 attenuant degrees. No alteration in expression, localization and morphology was observed when HEp‐Sm‐D1 or HEp‐2 interacted with the reference sera which could react with Ro/SSA, La/SSB, β2GP1, centromere, histone, and Scl‐70 antibodies in routine IIF tests.
Conclusion
As a new kind of substrate of IIF, HEp‐Sm‐D1 can be used to detect anti‐Sm antibodies. Transfected HEp‐2 cells keep the immunofluorescent property of HEp‐2 cells in immunofluorescence anti‐nuclear antibody (IFANA) test and could potentially be used as substrate for routine IFANA detection.</abstract><cop>England</cop><pub>Blackwell Publishing Ltd</pub><pmid>23981752</pmid><doi>10.1111/1756-185X.12000</doi><tpages>7</tpages></addata></record> |
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| ispartof | International journal of rheumatic diseases, 2013-06, Vol.16 (3), p.303-309 |
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| subjects | Antigen-Antibody Reactions antigenic localization Autoantibodies - blood Biomarkers - blood Blotting, Western Carcinoma - genetics Carcinoma - metabolism Case-Control Studies Cell Line, Tumor Cloning, Molecular Epitopes Green Fluorescent Proteins - genetics Green Fluorescent Proteins - metabolism HEp-2 cells Humans indirect immunofluorescent technique Laryngeal Neoplasms - genetics Laryngeal Neoplasms - metabolism Lupus Erythematosus, Systemic - blood Lupus Erythematosus, Systemic - diagnosis Lupus Erythematosus, Systemic - immunology Microscopy, Confocal Microscopy, Fluorescence Recombinant Fusion Proteins - genetics Recombinant Fusion Proteins - metabolism Reverse Transcriptase Polymerase Chain Reaction Sm-D1 snRNP Core Proteins - genetics snRNP Core Proteins - metabolism Transfection |
| title | Expression, localization and clinical application of exogenous Smith proteins D1 in gene transfected HEp-2 cells |
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