Quantitative expression patterns of peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) protein in mice

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Abstract

The expression patterns of PPARβ/δ have been described, but the majority of these data are based on mRNA data. To date, there are no reports that have quantitatively examined the expression of PPARβ/δ protein in mouse tissues. In the present study, a highly specific PPARβ/δ antibody was developed, characterized, and used to examine tissue expression patterns of PPARβ/δ. As compared to commercially available anti-PPARβ/δ antibodies, one of six polyclonal anti-PPARβ/δ antibodies developed was significantly more effective for immunoprecipitation of in vitro-translated PPARβ/δ. This antibody was used for quantitative Western blot analysis using radioactive detection methods. Expression of PPARβ/δ was highest in colon, small intestine, liver, and keratinocytes as compared to other tissues including heart, spleen, skeletal muscle, lung, brain, and thymus. Interestingly, PPARβ/δ expression was localized in the nucleus and RXRα can be co-immunoprecipitated with nuclear PPARβ/δ. Results from these studies demonstrate that PPARβ/δ expression is highest in intestinal epithelium, liver, and keratinocytes, consistent with significant biological roles in these tissues.

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Materials and methods

Anti-PPARβδ antibody characterization. Antiserum from six rabbits (designated 8095–8100) immunized with a PPARβ/δ-specific peptide (amino acids 1–29; sequence NP-035275) was obtained from Affinity Bioreagents. Antiserum was used to screen for PPARβ/δ immunoreactivity using Western blot analysis. COS-1 cells were transfected with a mouse PPARβ/δ expression vector, kindly provided by Drs. Walter Wahli and Pallavi Devchand, and cell lysate used for a positive control for these experiments. COS-1

Results

All six of antisera obtained from Affinity Bioreagents were immunoreactive with the COS-1 cell lysate with high PPARβ/δ expression (Fig. 1A). Of the six antisera examined, rabbit sera 8095, 8099, and 8100 were selected for further characterization after affinity purification. All three affinity purified antibodies immunoprecipitated in vitro-translated PPARβ/δ, with the percentage of immunoprecipitated protein ranging from 12–32% (Fig. 1B), which is quite efficient relative to other antibodies.

Discussion

Previous studies by others have described the expression patterns of PPARβ/δ mRNA in rat and mouse tissues with some variability noted. For example, one of the first studies that examined expression of PPARβ/δ mRNA by Northern blot analysis of rat samples noted the highest expression in adrenal gland, heart, and intestine, with modest expression in brain, kidney, and spleen, and low expression in liver and testis [10]. In mice, expression of PPARβ/δ mRNA was highest in mouse liver, with modest

Acknowledgments

Supported in part by the National Institutes of Health Grants CA97999 and CA124533 (J.M.P.).

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