Characterization of human 1,25-dihydroxyvitamin D3 receptor anti-peptide antibodies.
Rabbit and chicken antibodies were raised against two peptides synthesized according to the structure of human 1,25-dihydroxyvitamin D3 receptor (hVDR): rabbit alpha hVDR-103 against the N-terminal amino acids 5-18 and alpha hVDR-104 against the amino acids 172-186 in the hinge region and chicken alpha hVDR-cab11 against the amino acids 172-186, respectively. The specificity of the antibodies was tested by peptide saturation, SDS-PAGE immunoblotting, gel shift assay and sucrose gradient centrifugation. Immunoblotting of a soluble extract (cytosol) from osteosarcoma cell line MG-63 showed a single band with an M(r) of about 48,000 and human intestine cytosol a broad band (50-63,000) for both antibodies. The antibodies recognized activated (3.2S) hVDR by shifting the centrifugation sedimentation profile to 5-6S. The antibodies showed nuclear immunostaining of unoccupied VDR in human osteosarcoma cells MG-63, U2-Os and SaOs-2. The immunoreaction could be saturated with the corresponding synthetic peptide. In immunoblot alpha hVDR-103 reacted with human and rat VDR, whereas alpha hVDR-104 recognized human VDR only. Similarly in immunohistochemistry, alpha hVDR-103 showed staining with hVDR and rVDR, whereas alpha hVDR-104 reacted only with hVDR. All antibodies recognized the native hVDR as verified with sucrose gradient centrifugation or immunoprecipitation but only alpha hVDR-103 and alpha hVDR-cab11 in gel shift assay of hVDR associated with the vitamin D-responsive element of human osteocalcin gene promoter.
Tuohimaa
P
P
Department of Biomedical Sciences, University of Tampere, Finland.
Bläuer
M
M
Jääskeläinen
T
T
Itkonen
A
A
Lindfors
M
M
Mahonen
A
A
Palvimo
J
J
Vilja
P
P
Mäenpää
P H
PH
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry; D000918:Antibody Specificity; D002499:Centrifugation, Density Gradient; D002645:Chickens; D005260:Female; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008297:Male; D008969:Molecular Sequence Data; D012516:Osteosarcoma / Q000276:immunology; D010455:Peptides / Q000276:immunology; D011233:Precipitin Tests; D011817:Rabbits; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid / Q000276:immunology*; D014407:Tumor Cells, Cultured
C034373:1,24-dihydroxycholecalciferol receptor; D000906:Antibodies; D010455:Peptides; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid