Rainbow trout glucocorticoid receptor overexpression in Escherichia coli: production of antibodies for western blotting and immunohistochemistry.
Fragments of cDNA that encode the N-terminal and DNA-binding domains (DBD) of the rainbow trout glucocorticoid receptor (rtGR) were expressed in Escherichia coli as fusion proteins with glutathione-S-transferase (GST). The fusion proteins induced by IPTG could readily be detected as 45- and 40-kDa bands, respectively, in crude extracts, as well as in proteins purified on glutathione-agarose. These purified hybrid proteins were used to immunize rabbits. The antisera produced were tested for specificity by Western blot analysis using extracts from COS-1 cells transfected with an rtGR expression vector and from trout liver cells. The antisera raised against the DBD domain did not detect any bands on Western blots, even at low antiserum dilution. However, the purified DBD fusion protein specifically bound GRE-containing DNA fragments in gel-shift assays, and the retarded complexes were supershifted by these antibodies. The antisera raised against the N-terminal domain consistently detected two protein bands at 104 and 100 kDa in the two cell extracts and allowed specific immunohistochemical staining in fish brain and pituitary. For the first time in fish, these antibodies will allow analysis of GR expression in different cortisol target tissues.
Tujague
M
M
UPRES-A CNRS 6026, Endocrinologie Moléculaire des Poissons, INRA, Rennes, France.
Saligaut
D
D
Teitsma
C
C
Kah
O
O
Valotaire
Y
Y
Ducouret
B
B
D000818:Animals; D000906:Antibodies / Q000737:chemistry; D000917:Antibody Formation; D015153:Blotting, Western; D001923:Brain Chemistry / Q000502:physiology; D003600:Cytosol / Q000378:metabolism; D015342:DNA Probes; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005260:Female; D005982:Glutathione Transferase / Q000378:metabolism; D007150:Immunohistochemistry; D008099:Liver / Q000378:metabolism; D017686:Oncorhynchus mykiss / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011817:Rabbits / Q000276:immunology; D011965:Receptors, Glucocorticoid / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D000906:Antibodies; D015342:DNA Probes; D011965:Receptors, Glucocorticoid; D011994:Recombinant Proteins; D005982:Glutathione Transferase