Development and characterization of a panel of monoclonal antibodies against the novel subtilisin-like proprotein processing enzyme furin.
Monoclonal antibodies were raised against the recently discovered subtilisin-like proprotein processing enzyme furin. As immunogen, a bacterially expressed hybrid protein was used which consisted of glutathione S-transferase fused to almost the entire human furin protein. Ten monoclonal antibodies were obtained and these could be divided into four categories on the basis of their reactivity towards a number of bacterially expressed hybrid proteins, each of which contained a different portion of human furin. Four of the monoclonal antibodies did not recognize mouse furin. All monoclonal antibodies were tested for their applicability in Western blot and immunofluorescence analysis. Western blot analysis was performed with COS-1 cells in which biologically active forms of human and mouse furin were expressed transiently under control of the SV40 late promoter. This approach was necessary, since physiological levels of fur gene encoded proteins appeared to be very low. In cells transfected with human or mouse fur cDNA, a protein of about 100 kDa and a doublet of about 90 kDa could be detected with most of the monoclonal antibodies. Some of these antibodies appeared to be also reactive in immunofluorescence analysis of transfected COS-1 cells.
van Duijnhoven
H L
HL
Department of Biochemistry, University of Nijmegen, The Netherlands.
Creemers
J W
JW
Kranenborg
M G
MG
Timmer
E D
ED
Groeneveld
A
A
van den Ouweland
A M
AM
Roebroek
A J
AJ
van de Ven
W J
WJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D001666:Binding Sites, Antibody; D015139:Blotting, Southern; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005455:Fluorescent Antibody Technique; D045683:Furin; D006801:Humans; D007124:Immunoenzyme Techniques; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology; D013381:Subtilisins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D014162:Transfection
D000911:Antibodies, Monoclonal; D011993:Recombinant Fusion Proteins; D004247:DNA; D013381:Subtilisins; D045683:Furin