{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,28]],"date-time":"2026-04-28T07:38:18Z","timestamp":1777361898334,"version":"3.51.4"},"reference-count":49,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2024,6,1]],"date-time":"2024-06-01T00:00:00Z","timestamp":1717200000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100014943","name":"Sarepta Therapeutics","doi-asserted-by":"publisher","award":["444015-RC-57271"],"award-info":[{"award-number":["444015-RC-57271"]}],"id":[{"id":"10.13039\/100014943","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Sarcospan (SSPN) is a 25-kDa transmembrane protein that is broadly expressed at the cell surface of many tissues, including, but not limited to, the myofibers from skeletal and smooth muscles, cardiomyocytes, adipocytes, kidney epithelial cells, and neurons. SSPN is a core component of the dystrophin\u2013glycoprotein complex (DGC) that links the intracellular actin cytoskeleton with the extracellular matrix. It is also associated with integrin \u03b17\u03b21, the predominant integrin expressed in skeletal muscle. As a tetraspanin-like protein with four transmembrane spanning domains, SSPN functions as a scaffold to facilitate protein\u2013protein interactions at the cell membrane. Duchenne muscular dystrophy, Becker muscular dystrophy, and X-linked dilated cardiomyopathy are caused by the loss of dystrophin at the muscle cell surface and a concomitant loss of the entire DGC, including SSPN. SSPN overexpression ameliorates Duchenne muscular dystrophy in the mdx murine model, which supports SSPN being a viable therapeutic target. Other rescue studies support SSPN as a biomarker for the proper assembly and membrane expression of the DGC. Highly specific and robust antibodies to SSPN are needed for basic research on the molecular mechanisms of SSPN rescue, pre-clinical studies, and biomarker evaluations in human samples. The development of SSPN antibodies is challenged by the presence of its four transmembrane domains and limited antigenic epitopes. To address the significant barrier presented by limited commercially available antibodies, we aimed to generate a panel of robust SSPN-specific antibodies that can serve as a resource for the research community. We created antibodies to three SSPN protein epitopes, including the intracellular N- and C-termini as well as the large extracellular loop (LEL) between transmembrane domains 3 and 4. We developed a panel of rabbit antibodies (poly- and monoclonal) against an N-terminal peptide fragment of SSPN. We used several assays to show that the rabbit antibodies recognize mouse SSPN with a high functional affinity and specificity. We developed mouse monoclonal antibodies against the C-terminal peptide and the large extracellular loop of human SSPN. These antibodies are superior to commercially available antibodies and outperform them in various applications, including immunoblotting, indirect immunofluorescence analysis, immunoprecipitation, and an ELISA. These newly developed antibodies will significantly improve the quality and ease of SSPN detection for basic and translational research.<\/jats:p>","DOI":"10.3390\/ijms25116121","type":"journal-article","created":{"date-parts":[[2024,6,3]],"date-time":"2024-06-03T10:06:01Z","timestamp":1717409161000},"page":"6121","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["The Development of Robust Antibodies to Sarcospan, a Dystrophin- and Integrin-Associated Protein, for Basic and Translational Research"],"prefix":"10.3390","volume":"25","author":[{"given":"Ekaterina I.","family":"Mokhonova","sequence":"first","affiliation":[{"name":"Department of Integrative Biology and Physiology, University of California Los Angeles, Los Angeles, CA 90095, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ravinder","family":"Malik","sequence":"additional","affiliation":[{"name":"Department of Integrative Biology and Physiology, University of California Los Angeles, Los Angeles, CA 90095, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hafsa","family":"Mamsa","sequence":"additional","affiliation":[{"name":"Department of Integrative Biology and Physiology, University of California Los Angeles, Los Angeles, CA 90095, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jackson","family":"Walker","sequence":"additional","affiliation":[{"name":"Department of Integrative Biology and Physiology, University of California Los Angeles, Los Angeles, CA 90095, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elizabeth M.","family":"Gibbs","sequence":"additional","affiliation":[{"name":"Department of Integrative Biology and Physiology, University of California Los Angeles, Los Angeles, CA 90095, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rachelle H.","family":"Crosbie","sequence":"additional","affiliation":[{"name":"Department of Integrative Biology and Physiology, University of California Los Angeles, Los Angeles, CA 90095, USA"},{"name":"Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California Los Angeles, Los Angeles, CA 90095, USA"},{"name":"Molecular Biology Institute, University of California Los Angeles, Los Angeles, CA 90095, USA"},{"name":"Department of Neurology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2024,6,1]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/2044-5040-3-1","article-title":"Sarcospan: A small protein with large potential for Duchenne muscular dystrophy","volume":"3","author":"Marshall","year":"2013","journal-title":"Skelet. Muscle"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/s13395-022-00311-x","article-title":"Multi-omics analysis of sarcospan overexpression in mdx skeletal muscle reveals compensatory remodeling of cytoskeleton-matrix interactions that promote mechanotransduction pathways","volume":"13","author":"McCourt","year":"2023","journal-title":"Skelet. Muscle"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"31221","DOI":"10.1074\/jbc.272.50.31221","article-title":"Sarcospan, the 25-kDa transmembrane component of the dystrophin-glycoprotein complex","volume":"272","author":"Crosbie","year":"1997","journal-title":"J. Biol. Chem."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"153","DOI":"10.1083\/jcb.145.1.153","article-title":"Membrane targeting and stabilization of sarcospan is mediated by the sarcoglycan subcomplex","volume":"145","author":"Crosbie","year":"1999","journal-title":"J. Cell Biol."},{"key":"ref_5","first-page":"11","article-title":"Stabilization of the cardiac sarcolemma by sarcospan rescues DMD-associated cardiomyopathy","volume":"5","author":"Parvatiyar","year":"2019","journal-title":"JCI Insight"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"639","DOI":"10.1016\/j.yexcr.2006.11.021","article-title":"Structural and functional analysis of the sarcoglycan-sarcospan subcomplex","volume":"313","author":"Miller","year":"2007","journal-title":"Exp. Cell Res."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"349","DOI":"10.1016\/S0959-437X(02)00309-X","article-title":"Muscular dystrophies involving the dystrophin-glycoprotein complex: An overview of current mouse models","volume":"12","author":"Durbeej","year":"2002","journal-title":"Curr. Opin. Genet. Dev."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2019","DOI":"10.1093\/hmg\/9.13.2019","article-title":"Molecular and genetic characterization of sarcospan: Insights into sarcoglycan-sarcospan interactions","volume":"9","author":"Crosbie","year":"2000","journal-title":"Hum. Mol. Genet."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1016\/S1097-2765(00)80410-4","article-title":"Disruption of the beta-sarcoglycan gene reveals pathogenetic complexity of limb-girdle muscular dystrophy type 2E","volume":"5","author":"Durbeej","year":"2000","journal-title":"Mol. Cell"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"465","DOI":"10.1016\/S0092-8674(00)81975-3","article-title":"Disruption of the sarcoglycan-sarcospan complex in vascular smooth muscle: A novel mechanism for cardiomyopathy and muscular dystrophy","volume":"98","author":"Cohn","year":"1999","journal-title":"Cell"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1461","DOI":"10.1083\/jcb.142.6.1461","article-title":"Progressive muscular dystrophy in alpha-sarcoglycan-deficient mice","volume":"142","author":"Duclos","year":"1998","journal-title":"J. Cell Biol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"419","DOI":"10.1083\/jcb.200808027","article-title":"Sarcospan reduces dystrophic pathology: Stabilization of the utrophin-glycoprotein complex","volume":"183","author":"Peter","year":"2008","journal-title":"J. Cell Biol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1009","DOI":"10.1083\/jcb.201110032","article-title":"Sarcospan-dependent Akt activation is required for utrophin expression and muscle regeneration","volume":"197","author":"Marshall","year":"2012","journal-title":"J. Cell Biol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"2011","DOI":"10.1093\/hmg\/ddu615","article-title":"Sarcospan integration into laminin-binding adhesion complexes that ameliorate muscular dystrophy requires utrophin and alpha7 integrin","volume":"24","author":"Marshall","year":"2015","journal-title":"Hum. Mol. Genet."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"718","DOI":"10.1093\/hmg\/ddab276","article-title":"Sarcospan increases laminin-binding capacity of alpha-dystroglycan to ameliorate DMD independent of Galgt2","volume":"31","author":"Mamsa","year":"2022","journal-title":"Hum. Mol. Genet."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"4378","DOI":"10.1093\/hmg\/dds271","article-title":"Dystrophin and utrophin expression require sarcospan: Loss of alpha7 integrin exacerbates a newly discovered muscle phenotype in sarcospan-null mice","volume":"21","author":"Marshall","year":"2012","journal-title":"Hum. Mol. Genet."},{"key":"ref_17","first-page":"5395","article-title":"High levels of sarcospan are well tolerated and act as a sarcolemmal stabilizer to address skeletal muscle and pulmonary dysfunction in DMD","volume":"25","author":"Gibbs","year":"2016","journal-title":"Hum. Mol. Genet."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"e002481","DOI":"10.1161\/JAHA.115.002481","article-title":"Sarcospan Regulates Cardiac Isoproterenol Response and Prevents Duchenne Muscular Dystrophy-Associated Cardiomyopathy","volume":"4","author":"Parvatiyar","year":"2015","journal-title":"J. Am. Heart Assoc."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"996","DOI":"10.1242\/jcs.03360","article-title":"Disrupted mechanical stability of the dystrophin-glycoprotein complex causes severe muscular dystrophy in sarcospan transgenic mice","volume":"120","author":"Peter","year":"2007","journal-title":"J. Cell Sci."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"32","DOI":"10.1186\/s13395-019-0218-x","article-title":"Development of a high-throughput screen to identify small molecule enhancers of sarcospan for the treatment of Duchenne muscular dystrophy","volume":"9","author":"Shu","year":"2019","journal-title":"Skelet. Muscle"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"26","DOI":"10.1186\/s13395-020-00244-3","article-title":"High-throughput screening identifies modulators of sarcospan that stabilize muscle cells and exhibit activity in the mouse model of Duchenne muscular dystrophy","volume":"10","author":"Shu","year":"2020","journal-title":"Skelet. Muscle"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"479","DOI":"10.1007\/978-1-0716-2772-3_25","article-title":"High-Throughput Screening to Identify Modulators of Sarcospan","volume":"2587","author":"Shu","year":"2023","journal-title":"Methods Mol. Biol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1669","DOI":"10.1128\/MCB.20.5.1669-1677.2000","article-title":"Sarcospan-deficient mice maintain normal muscle function","volume":"20","author":"Lebakken","year":"2000","journal-title":"Mol. Cell Biol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1143","DOI":"10.1016\/j.it.2021.10.008","article-title":"Single B cell technologies for monoclonal antibody discovery","volume":"42","author":"Pedrioli","year":"2021","journal-title":"Trends Immunol."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Starkie, D.O., Compson, J.E., Rapecki, S., and Lightwood, D.J. (2016). Generation of Recombinant Monoclonal Antibodies from Immunised Mice and Rabbits via Flow Cytometry and Sorting of Antigen-Specific IgG+ Memory B Cells. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0152282"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Lei, L., Tran, K., Wang, Y., Steinhardt, J.J., Xiao, Y., Chiang, C.I., Wyatt, R.T., and Li, Y. (2019). Antigen-Specific Single B Cell Sorting and Monoclonal Antibody Cloning in Guinea Pigs. Front. Microbiol., 10.","DOI":"10.3389\/fmicb.2019.00672"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"326","DOI":"10.1016\/j.ygeno.2007.12.005","article-title":"Appearance of new tetraspanin genes during vertebrate evolution","volume":"91","author":"Chung","year":"2008","journal-title":"Genomics"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"801","DOI":"10.1038\/nrm1736","article-title":"Tetraspanin functions and associated microdomains","volume":"6","author":"Hemler","year":"2005","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"136","DOI":"10.1038\/nri1548","article-title":"The tetraspanin web modulates immune-signalling complexes","volume":"5","author":"Levy","year":"2005","journal-title":"Nat. Rev. Immunol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"434","DOI":"10.1016\/j.tcb.2009.06.004","article-title":"Tetraspanin-enriched microdomains: A functional unit in cell plasma membranes","volume":"19","author":"Barreiro","year":"2009","journal-title":"Trends Cell Biol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"106","DOI":"10.1016\/S0968-0004(02)00014-2","article-title":"Functional domains in tetraspanin proteins","volume":"28","author":"Stipp","year":"2003","journal-title":"Trends Biochem. Sci."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"218","DOI":"10.1152\/physiol.00015.2005","article-title":"Protein-protein interactions in the tetraspanin web","volume":"20","author":"Levy","year":"2005","journal-title":"Physiology"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"741","DOI":"10.1042\/BST20160284","article-title":"Molecular interactions shaping the tetraspanin web","volume":"45","author":"Dunlock","year":"2017","journal-title":"Biochem. Soc. Trans."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1103","DOI":"10.1083\/jcb.200108061","article-title":"Specific tetraspanin functions","volume":"155","author":"Hemler","year":"2001","journal-title":"J. Cell Biol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"12","DOI":"10.1093\/emboj\/20.1.12","article-title":"CD81 extracellular domain 3D structure: Insight into the tetraspanin superfamily structural motifs","volume":"20","author":"Kitadokoro","year":"2001","journal-title":"EMBO J."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"40055","DOI":"10.1074\/jbc.M105557200","article-title":"Structure of the tetraspanin main extracellular domain. A partially conserved fold with a structurally variable domain insertion","volume":"276","author":"Seigneuret","year":"2001","journal-title":"J. Biol. Chem."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"25005","DOI":"10.1074\/jbc.M102156200","article-title":"Transmembrane-4 superfamily proteins associate with activated protein kinase C (PKC) and link PKC to specific beta(1) integrins","volume":"276","author":"Zhang","year":"2001","journal-title":"J. Biol. Chem."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"261","DOI":"10.1091\/mbc.e12-09-0642","article-title":"CD81 regulates cell migration through its association with Rac GTPase","volume":"24","author":"Tejera","year":"2013","journal-title":"Mol. Biol. Cell"},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Marshall, J.L., Kwok, Y., McMorran, B.J., Baum, L.G., and Crosbie-Watson, R.H. (2013). The potential of sarcospan in adhesion complex replacement therapeutics for the treatment of muscular dystrophy. FEBS J.","DOI":"10.1111\/febs.12295"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"735","DOI":"10.1080\/19420862.2017.1325052","article-title":"Antibodies targeting G protein-coupled receptors: Recent advances and therapeutic challenges","volume":"9","author":"Ayoub","year":"2017","journal-title":"MAbs"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1186\/s13395-017-0127-9","article-title":"Nanospan, an alternatively spliced isoform of sarcospan, localizes to the sarcoplasmic reticulum in skeletal muscle and is absent in limb girdle muscular dystrophy 2F","volume":"7","author":"Peter","year":"2017","journal-title":"Skelet. Muscle"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"747","DOI":"10.1038\/nrd2659","article-title":"Targeting of tetraspanin proteins--potential benefits and strategies","volume":"7","author":"Hemler","year":"2008","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"545","DOI":"10.1007\/s10974-006-9069-2","article-title":"Over-expression of Microspan, a novel component of the sarcoplasmic reticulum, causes severe muscle pathology with triad abnormalities","volume":"27","author":"Miller","year":"2006","journal-title":"J. Muscle Res. Cell Motil."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"5320","DOI":"10.18632\/oncotarget.2097","article-title":"Functional genomics identifies novel genes essential for clear cell renal cell carcinoma tumor cell proliferation and migration","volume":"5","author":"Marlow","year":"2014","journal-title":"Oncotarget"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"2","DOI":"10.1186\/1745-7580-2-2","article-title":"Improved method for predicting linear B-cell epitopes","volume":"2","author":"Larsen","year":"2006","journal-title":"Immunome Res."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"1088","DOI":"10.1038\/nm.1966","article-title":"A rapid and efficient single-cell manipulation method for screening antigen-specific antibody-secreting cells from human peripheral blood","volume":"15","author":"Jin","year":"2009","journal-title":"Nat. Med."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"143","DOI":"10.4161\/mabs.27044","article-title":"The rapid generation of recombinant functional monoclonal antibodies from individual, antigen-specific bone marrow-derived plasma cells isolated using a novel fluorescence-based method","volume":"6","author":"Clargo","year":"2014","journal-title":"MAbs"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"495","DOI":"10.1038\/256495a0","article-title":"Continuous cultures of fused cells secreting antibody of predefined specificity","volume":"256","author":"Kohler","year":"1975","journal-title":"Nature"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"17792","DOI":"10.1038\/s41598-022-20560-6","article-title":"Highly efficient hybridoma generation and screening strategy for anti-PD-1 monoclonal antibody development","volume":"12","author":"Phakham","year":"2022","journal-title":"Sci. Rep."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/11\/6121\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T14:52:21Z","timestamp":1760107941000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/11\/6121"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,6,1]]},"references-count":49,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2024,6]]}},"alternative-id":["ijms25116121"],"URL":"https:\/\/doi.org\/10.3390\/ijms25116121","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,6,1]]}}}