Abstract
In an effort to understand the relationship between a 72-kDa heat shock protein (Hsp72) and peroxisome proliferator-activated receptors (PPARs), we have characterized their interaction using clofibric acid-Sepharose chromatography and co-immunoprecipitation with antisera raised against either rat PPAR (rPPAR) or Hsp72. First, we observed that both rPPAR and Hsp72 elute in a clofibrate-dependent manner from the clofibric acid-Sepharose matrix. Second, we found that immunoprecipitation of either protein from solution resulted in the precipitation of the other. This result was obtained from rat liver cytosol, from Spodoptera frugiperda (Sf9) insect cells expressing rPPAR, and from reticulocyte lysate rPPAR expression systems. These results suggest that Hsp72 and rPPAR form a complex in vivo and that Hsp72 may play a role in the folding, subcellular localization, and/or signaling pathway of PPARs.
Highlights
72-kDa heat shock protein (Hsp72) and peroxisome pro-exist (5)
Liferator-activated receptors (PPARs), we have charac- encoding a novel member of steroid hormone receptor superterized their interactionusing clofibric acid-Sepharose family was shown to be required for PP-induced expression of chromatography and co-immunoprecipitation withan- reporter genes linked to elements present in the 5”flanking tisera raised againsteither rat peroxisome proliferator-activated receptor (PPAR) or Hsp72. region of the bifunctional enoyl-CoA hydratasel3-hydroxyacyl
After overnight incubatioant 4 "C, antigen-antibody complexes were collected by the addition of 50 p1 of 50% protein A-agarose (Sigma) and the immune complexes washed four times with 500 pI of RIPA buffer (10 mM Tris-HCI, pH 8.0, 150 mM NaCI, 1% Triton X-100, 1%sodiumdeoxycholate).After the finalwash, the complexes were react with this protein (F2igB., lane 1), the results suggest that PPAR from rat liver cytosol is retained on the clofibric-acid Sepharose
Summary
72-kDa heat shock protein (Hsp72) and peroxisome pro-exist (5). Using transient expression systems, a mouse cDNA liferator-activated receptors (PPARs), we have charac- encoding a novel member of steroid hormone receptor superterized their interactionusing clofibric acid-Sepharose family was shown to be required for PP-induced expression of chromatography and co-immunoprecipitation withan- reporter genes linked to elements present in the 5”flanking tisera raised againsteither rat PPAR (rPPAR) or Hsp72. region of the bifunctional enoyl-CoA hydratasel3-hydroxyacyl-. Binding of rPPAR to Clofibric Acid-Sepharose Affinity Matrix-SDS-PAGE of rat liver proteins that bound to clofibric acid-Sepharoseyielded a major72-kDaproteinandminor bands migratingat 55 (Fig. 2 A ) and 31 kDa (14).
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