Characterisation of the transcription factor, SIX5, using a new panel of monoclonal antibodies.
SIX5 is a member of the human SIX family of transcription factors, many of which are involved in eye development. However, SIX5 transcripts are known to be present at very low levels in cells and no study has yet convincingly demonstrated detection of endogenous SIX5 protein by Western blotting or immunolocalisation. We have produced a new panel of 18 monoclonal antibodies (mAbs) that recognise at least four different epitopes in order to identify authentic human SIX5 protein in cells and tissues. Phage-displayed peptide libraries were used to identify individual amino-acids important for antibody binding within each epitope. Endogenous SIX5 migrated in SDS-PAGE with an apparent M(r) of 100 kDa and was present at similar levels in all foetal tissues and cell lines tested. In HeLa cells, it was located in the nucleoplasm with a granular distribution. An mRNA for a shorter splicing isoform of SIX5, with an altered carboxy-terminus, has been described, but further mAbs specific for this isoform did not detect any endogenous protein. We conclude that the full-length isoform is the major functional protein in vivo while the putative shorter protein is undetectable and may not be expressed at all.
Pham
Y Chan N
YC
Biochemistry Group, North East Wales Institute, Mold Road, Wrexham, United Kingdom.
Man
Nguyen thi
Nt
Holt
Ian
I
Sewry
Caroline A
CA
Pall
Gurman
G
Johnson
Keith
K
Morris
Glenn E
GE
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology*; D005333:Fetus; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D006825:Hybridomas; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D019151:Peptide Library; D020033:Protein Isoforms; D012333:RNA, Messenger; D017386:Sequence Homology, Amino Acid
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D000939:Epitopes; D018398:Homeodomain Proteins; D019151:Peptide Library; D020033:Protein Isoforms; D012333:RNA, Messenger; C106687:SIX5 protein, human