title: Generation and characterization of mouse monoclonal antibody 5E1 against human transcription factor GLI3.
abstract: GLI3 is a transcriptional effector of the developmentally important hedgehog (Hh) signaling pathway. Here we report the production of mouse monoclonal antibody (MAb) against putative repressive motif in GLI3 (GLI3pRM). BALB/c mice were immunized with purified recombinant human GLI3pRM protein; the splenocytes from these mice were fused with myeloma cell line (SP2/0) by using standard hybridoma production techniques. Resulting hybridomas producing anti-GLI3pRM antibodies were screened by enzyme-linked immunosorbent assay (ELISA) and isotyped. The specificity of MAb 5E1 was determined based on its activity in Western blot and immunofluorescence analysis of human NT2/D1 cell line. The results showed that MAb 5E1 was immunoglobulin IgM/ê and it recognized recombinant human GLI3pRM specifically. In addition, MAb 5E1 bound to the full-length (FL-GLI3) as well as a short protein (GLI3R) and did not cross-react with a similar region in GLI2. MAb 5E1 could also be used to detect the expression of GLI3 in mouse cell lines and embryonic tissues.
authors.affiliation: Institute of Gene Technology, Tallinn University of Technology, Akadeemia tee, Tallinn, Estonia.
authors.forename: Reet
authors.initials: R
authors.lastname: Hunt
authors.forename: Olga
authors.initials: O
authors.lastname: Bragina
authors.forename: Monika
authors.initials: M
authors.lastname: Drews
authors.forename: Lagle
authors.initials: L
authors.lastname: Kasak
authors.forename: Sirje
authors.initials: S
authors.lastname: Timmusk
authors.forename: Andres
authors.initials: A
authors.lastname: Valkna
authors.forename: Priit
authors.initials: P
authors.lastname: Kogerman
authors.forename: Lilian
authors.initials: L
authors.lastname: Järvekülg
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D000918:Antibody Specificity; D001483:Base Sequence; D002460:Cell Line; D003429:Cross Reactions; D017931:DNA Primers / Q000235:genetics; D005260:Female; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007075:Immunoglobulin M / Q000096:biosynthesis; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D051741:Kruppel-Like Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D000074290:Zinc Finger Protein Gli3
chemical_list: D000911:Antibodies, Monoclonal; D017931:DNA Primers; C065683:GLI3 protein, human; C465571:Gli3 protein, mouse; D007075:Immunoglobulin M; D051741:Kruppel-Like Transcription Factors; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D000074290:Zinc Finger Protein Gli3