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18S was used as the reference gene, and relative gene expression was calculated using the method. Gene-specific primer sequences (5��-3��) are provided in Table 1. Immunoblot analysis was performed on RP adipose tissue samples to confirm observations at the mRNA level. Briefly, RP samples were homogenized in lysis buffer (50 mm Tris�CHCl, 0.1 mm EDTA, 0.1 mm EGTA, 0.1% mercaptoethanol, 1 mm phenylmethylsulfonyl fluoride, 2 ��m leupeptin, 1 ��m pepstatin and 20 mm CHAPS, pH 7.4) C59 wnt manufacturer using a tissue homogenizer (TissueLyser LT; Qiagen), and the total protein content from RP samples was measured using the Bradford method. Samples were then diluted with Laemmli buffer, and 5 ��g of protein from each sample was loaded onto polyacrylamide gels for separation by electrophoresis. Proteins were transferred to polyvinylidene difluoride membranes and probed with rabbit polyclonal antibodies NPR1 (1:125 dilution; Abcam, Cambridge, MA, USA), ��AR2 (1:500 dilution) and ��AR3 (1:100 dilution). Blots were reprobed for GAPDH (1:2500 dilution; Cell Signaling, Danvers, MA, USA). Band MAO intensities were quantified using ImageJ software (ImageJ, http://rsbweb.nih.gov/ij/), and values of target proteins were normalized to the intensity for GAPDH. Commercial antibodies for NPR2 were unsuccessful in Western blot analyses. Differences among SED, CR and RUN groups were analysed using a one-way ANOVA and Fisher's LSD post hoc tests. Data from the adipose tissue organ culture experiments were analysed using a three-factor ANOVA (adrenaline �� ANP �� SNAP), with each factor having two levels (treated or not). Post hoc analysis was performed using Dunnet's test to determine differences between individual treatment conditions and the control conditions (i.e. no treatments). Statistical significance was accepted at P�� 0.05, and a P value between 0.05 and 0.10 was regarded as a trend. By design, body weight, percentage body fat and fat pad masses did not differ between RUN and CR animals, but each were significantly lower compared with SED animals (P MAPK inhibitor Fig. 1A�CD). As reported previously (Mikus et al. 2010), rats with access to running wheels increased daily running distance between weeks 4 (3.9 �� 0.2 km day?1) and 10 (10.9 �� 4 km day?1), after which running distance gradually declined to 3.6 �� 0.2 km day?1 at 40 weeks. Compared with SED, RP depots of RUN rats exhibited approximately two- to threefold greater mRNA levels of NPR1, NPR2, ��AR2 and ��AR3 (all P