title: Generation and characterization of monoclonal antibodies against tuberous sclerosis complex 2.
abstract: TSC1 and TSC2 are two recently identified tumor suppressor genes encoding hamartin and tuberin, respectively. They have been implicated in the pathogenesis of tuberous sclerosis, a neurological disorder linked with the development of hamartomas in numerous organs, including the brain, kidneys, heart, and liver. Both protein products of TSC1 and TSC2 form an intracellular complex exerting GTPase-activating (GAP) activity towards a small G protein Rheb (Ras homologue enriched in brain). Inhibition of Rheb is important for the positive regulation of mTOR pathway, while mutations of hamartin or tuberin result in uncontrolled cell cycle progression. Although the precise role for the TSC1/2 complex in tumor suppression is not clear, many studies have established a link with the regulation of transcription and protein biosynthesis, increasing susceptibility to apoptosis, cell differentiation, and cell cycle control. We describe the development of a monoclonal antibody specific towards TSC2/tuberin and characterize the suitability for Western blotting, immunoprecipitation, and immunofluorescent applications. The C-terminal region of TSC2 was expressed as a His-tag fusion protein in bacteria, affinity purified and used as an immunogen. Hybrid myelomas were produced from the spleenocytes of immunized mice and SP2/0 myeloma cells. Testing the specificity of cell culture supernatants from generated hybridomas towards recombinant His-TSC2C in ELISA assay allowed us to isolate a panel of positive clones. Further analysis of selected clones by Western blotting and immunoprecipitation revealed one clone, termed D6, which specifically recognized recombinant and endogenous TSC2. The specificity of generated antibody was also confirmed in TSC2(/) and TSC2(+/+) mouse embryo fibroblasts. In summary, the produced antibody is a useful tool in our research program and will be available for researchers investigating signal transduction pathways involving TSC1/2 signaling under physiological conditions and in human pathologies.
authors.affiliation: Kyiv National Taras Shevchenko University, Biological Faculty, Kyiv, Ukraine.
authors.forename: Oksana
authors.initials: O
authors.lastname: Malanchuk
authors.forename: Sergiy
authors.initials: S
authors.lastname: Palchevskyy
authors.forename: Galina
authors.initials: G
authors.lastname: Ovcharenko
authors.forename: Jodie
authors.initials: J
authors.lastname: Gwalter
authors.forename: Volodymyr
authors.initials: V
authors.lastname: Pozur
authors.forename: Ivan
authors.initials: I
authors.lastname: Gout
authors.forename: Valeriy
authors.initials: V
authors.lastname: Filonenko
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry* / Q000276:immunology; D000918:Antibody Specificity; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006825:Hybridomas; D051379:Mice; D008807:Mice, Inbred BALB C; D051381:Rats; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; C000624653:TSC2 protein, human; C000624654:Tsc2 protein, mouse; C000624655:Tsc2 protein, rat; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins