Quantitative expression patterns of peroxisome proliferator-activated receptor-beta/delta (PPARbeta/delta) protein in mice

Abstract

The expression patterns of PPARbeta/delta 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 PPARbeta/delta protein in mouse tissues. In the present study, a highly specific PPARbeta/delta antibody was developed, characterized, and used to examine tissue expression patterns of PPARbeta/delta. As compared to commercially available anti-PPARbeta/delta antibodies, one of six polyclonal anti-PPARbeta/delta antibodies developed was significantly more effective for immunoprecipitation of in vitro-translated PPARbeta/delta. This antibody was used for quantitative Western blot analysis using radioactive detection methods. Expression of PPARbeta/delta was highest in colon, small intestine, liver, and keratinocytes as compared to other tissues including heart, spleen, skeletal muscle, lung, brain, and thymus. Interestingly, PPARbeta/delta expression was localized in the nucleus and RXRalpha can be co-immunoprecipitated with nuclear PPARbeta/delta. Results from these studies demonstrate that PPARbeta/delta expression is highest in intestinal epithelium, liver, and keratinocytes, consistent with significant biological roles in these tissues.

Fig. 1

Characterization of specific anti-PPARβ/δ antibody. (A) Western blot analysis using the six different anti-sera. Lysate from COS-1 cells transfected with a mouse PPARβ/δ expression vector was used as a positive control. (B) Immunoprecipitation of in vitro translated PPARβ/δ using three different affinity purified anti-PPARβ/δ antibodies. % IP = percentage of immunoprecipitation based on the 10% input. (C) Western blot analysis of immunoprecipitated PPARβ/δ. Lysate from COS-1 cells transfected with a mouse PPARβ/δ expression vector was used for the first lane (COS-1), in vitro translated PPARβ/δ was used for the second lane (in vitro) and soluble protein from mouse primary keratinocytes was used for the third lane (keratinocyte) for each of the three different affinity purified antibodies (8095, 8099 and 8100). Immunoprecipitated protein was detected by western blot analysis as described in Materials and Methods. (D) Comparison of immunoprecipitation of several commercially available anti- PPARβ/δ antibodies. Affinity purified 8099 anti-PPARβ/δ antibody was compared with Affinity Bioreagents anti-PPARβ/δ antibody (ABR PA1–828), Santa Cruz anti-PPARβ/δ antibody (sc-7197) or Santa Cruz anti-PPARβ/δ antibody (sc-1987) for their relative ability to immunoprecipitate using ³⁵S-labeled in vitro translated PPARβ/δ.

Fig. 2

Quantitative expression of PPARβ/δ in male mouse tissues. Crude protein samples were prepared and analyzed by quantitative western blot analysis as described in Materials and Methods. Hybridization signals for PPARβ/δ protein was normalized to LDH and the normalized values are presented as the mean ± S.E.M. below the tissue samples. + = lysate from COS-1 cells transfected with a mouse PPARβ/δ expression vector as a positive control.

Fig. 3

Nuclear and cytosolic localization of PPARβ/δ in male mouse tissues. (A) Nuclear and cytosolic protein preparations were prepared and analyzed by quantitative western blot analysis as described in Materials and Methods. + = lysate from COS-1 cells transfected with a mouse PPARβ/δ expression vector as a positive control. Blots were probed with anti-lamin antibody to demonstrate enriched nuclear fractions and anti-LDH to demonstrate enriched cytosolic fractions. (B) Immunoprecipitation of PPARβ/δ was performed using the 8099 anti-PPARβ/δ antibody or IgG control and nuclear extract from liver and then separated by SDS-PAGE. Immunoblots were then probed with either the 8099 anti-PPARβ/δ antibody or RXRα. Digital light units (DLU) for each immunoprecipitation (PPARβ/δ or RXRα) were corrected for background and non-specific binding from the IgG control.

Fig. 4

Lack of PPARβ/δ expression in PPARβ/δ-null mouse small intestine. Nuclear and cytosolic protein preparations from small intestine were prepared from wild-type and PPARβ/δ-null mice and analyzed by quantitative western blot analysis as described in Materials and Methods. + = lysate from COS-1 cells transfected with a mouse PPARβ/δ expression vector as a positive control. Blots were probed with anti-lamin antibody to demonstrate enriched nuclear fractions and anti-LDH to demonstrate enriched cytosolic fractions.