Generation and characterization of mouse monoclonal antibody 5E1 against human transcription factor GLI3.
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.
Hunt
Reet
R
Institute of Gene Technology, Tallinn University of Technology, Akadeemia tee 15, Tallinn, Estonia.
Bragina
Olga
O
Drews
Monika
M
Kasak
Lagle
L
Timmusk
Sirje
S
Valkna
Andres
A
Kogerman
Priit
P
Järvekülg
Lilian
L
D020816:Amino Acid Motifs; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D000918:Antibody Specificity; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006825:Hybridomas; D007150:Immunohistochemistry; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000276:immunology*; D051379:Mice; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000276:immunology*; D010455:Peptides / Q000235:genetics / Q000276:immunology; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D000074290:Zinc Finger Protein Gli3
D000911:Antibodies, Monoclonal; C065683:GLI3 protein, human; D051741:Kruppel-Like Transcription Factors; D009419:Nerve Tissue Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D000074290:Zinc Finger Protein Gli3