Monoclonal antibodies with selective specificity towards different glycosylation isoforms of FGFR1.
Fibroblast growth factor receptor 1 (FGFR1) is a member of the FGFR family of receptor tyrosine kinases, whose function has been implicated in diverse biological processes including cell proliferation, differentiation, survival, and tumorigenesis. This diversity is possibly mediated by the existence of multiple FGFR1 isoforms, generated by alternative splicing and post-translational modifications, mainly through glycosylation. In this study we report the generation and characterization of a panel of monoclonal antibodies directed towards FGFR1. To achieve this, we used as an antigen a fragment of FGFR1, corresponding to loop II-III of the extracellular domain, which shares low homology to other members of the FGFR family and possesses numerous antigentic determinants. Two rounds of ELISA screening and Western blot analysis allowed us to isolate a panel of monoclonal antibodies, which recognize specifically recombinant FGFR1 loop II-III. The ability of generated antibodies to recognize endogenous FGFR1 was examined in 3T3 L1 cells, which are known to express FGFR1, but not other members of FGFR family. Immunoblot analysis of 3T3 L1 cell lysates with hybridoma media of selected clones revealed a different, but overlapping pattern of immunoreactive bands, which might represent splicing and post-translationally modified forms of FGFR1. Furthermore, we also tested the cross-reactivity of generated antibodies towards recombinant full-length FGFR3 and their ability to recognize FGFR1 in 3T3 L1 cells by cyto- and immunocytochemistry. In summary, generated antibodies should be useful as tools for examining the expression pattern and biological functions of FGFR1 in normal and pathological tissues.
Gorbenko
Olena
O
Department of Cell Signalling, Institute of Molecular Biology and Genetics, NAS of Ukraine, Kyiv, Ukraine.
Ovcharenko
Galina
G
Volkova
Darija
D
Mayilo
Dmytro
D
Gaman
Nadia
N
Khozhayenko
Yulia
Y
Usenko
Valeriy
V
Gout
Ivan
I
Filonenko
Valeriy
V
D041721:3T3-L1 Cells; D017398:Alternative Splicing; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002454:Cell Differentiation; D049109:Cell Proliferation; D002470:Cell Survival; D005260:Female; D005786:Gene Expression Regulation; D006031:Glycosylation; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D017434:Protein Structure, Tertiary; D051496:Receptor, Fibroblast Growth Factor, Type 1 / Q000737:chemistry*
D000911:Antibodies, Monoclonal; C496348:FGFR1 protein, human; D051496:Receptor, Fibroblast Growth Factor, Type 1