RESUMEN
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, and 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 analyses of the human NT2/D1 cell line. The results showed that MAb 5E1 was immunoglobulin IgM/kappa, recognizing 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.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/química , Factores de Transcripción de Tipo Kruppel/inmunología , Proteínas del Tejido Nervioso/inmunología , Secuencias de Aminoácidos , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Especificidad de Anticuerpos , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Humanos , Hibridomas , Inmunohistoquímica , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Proteínas del Tejido Nervioso/genética , Péptidos/genética , Péptidos/inmunología , Pliegue de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteína Gli3 con Dedos de ZincRESUMEN
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.