Nuclear localization and hepatic zonation of rat "spot 14" protein: immunohistochemical investigation employing anti-fusion protein antibodies.
S14 protein and mRNA levels are rapidly regulated by hormones and diet. We have purified a 45-Kd fusion protein from lysates of transformed E. coli that includes the entire S14 polypeptide. Affinity-purified rabbit anti-fusion protein antibodies were used in immunohistochemistry to determine the distribution of S14 protein across the hepatic lobule, and to reassess its intracellular location. In hyperthyroid liver, S14 protein clustered near the central venous zone, and was not detectable in the periportal area of the acinus. The signal in perivenous hepatocytes was primarily nuclear in location, in stark contrast to previous subcellular fractionation studies. Visualization of identical hepatic distribution and subcellular localization employing anti-synthetic peptide antiserum provided evidence for the specificity of the immunostaining, as did attenuation of the signal by preincubation of the antibody with its antigen. No staining was observed in sections of heart or hypothyroid liver, as expected from the low levels of S14 protein in those tissues. The data indicate that induction of S14 protein expression by T3 occurs through enhanced expression by perivenous hepatocytes, rather than by recruitment of cells in more peripheral zones of the lobule. Nuclear localization of the S14 protein by immunohistochemistry suggests that it is lost from nuclei during standard fractionation procedures, and prompts consideration of a role for S14 in regulation of nuclear structure and/or function.
Kinlaw
W B
WB
Department of Medicine, Dartmouth Medical School, Lebanon, NH 03756.
Tron
P
P
Friedmann
A S
AS
D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D002467:Cell Nucleus / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics; D007150:Immunohistochemistry; D008099:Liver / Q000737:chemistry* / Q000648:ultrastructure; D008297:Male; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D011506:Proteins / Q000032:analysis* / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000276:immunology*; D014018:Tissue Distribution; D014157:Transcription Factors
D000906:Antibodies; D009687:Nuclear Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; C051065:THRSP protein, human; C501478:Thrsp protein, rat; D014157:Transcription Factors; D005982:Glutathione Transferase