Figure 5A Representative H&E histological sections of sWAT from WT and KO mice probed with anti-UCP1 antibodies. Mice were either maintained at 24°C (RT) or housed at 6°C for 5 days. Scale bar = 200 μm. Transcript levels of Pgc1α and Ucp1 in sWAT of male and female WT and KO mice after 24 hours at RT or 4°C (n= Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD. * = p < 0.05; ** = p < 0.01. (two-way ANOVA with multiple comparisons C Transcript levels of Ucp1, Pgc1α, Adrb3, retinoblastoma 1 (Rb1), zinc finger protein 423 (Zfp423), and PR domain containing 16 (Prdm16) were quantified in sWAT of WT and KO mice after 25 weeks on a HFD (n=4-5). Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD. * = p < 0.05; ** = p < 0.01. ; unpaired student's t-test D Transcript levels of Pgc1α-1b and Pgc1α-1a isoforms in sWAT of WT and KO 8 week old females maintained at 24°C (RT) or housed at 4°C for 24 hours (n=5). Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD. * = p < 0.05; ** = p < 0.01. (two-way ANOVA with multiple comparisons E. Western blot for RCAN1 in various skeletal muscle groups. Each lane was loaded with 20 μg of total protein from plantaris (PL), soleus (SOL), extensor digitorum longus (EDL), tibialis anterior (TA), red gastrocnemius (RG), white gastrocnemius (WG), white vastus lateralis (WV), heart ventricle (HT), atria, diaphragm (Dia), and Tong. The lane containing brain extract was loaded with 10 μg of protein. Antibody specificity was validated by running 20 μg of protein from the heart of an Rcan1-KO animal (HT-KO). The asterisks * indicate the location of putative proteolytic fragments of RCAN1.1. F Transcript levels of Sln in skeletal muscle (tibialis anterior) of 8-10 week old WT and KO mice housed at RT. (n=4-5 each gender). G Transcript levels of Sln in skeletal muscle (tibialis anterior ) of 8-10 week old male WT and KO animals housed at RT. (n=5).Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD. * = p < 0.05; ** = p < 0.01. (two-way ANOVA with multiple comparisons F, ; unpaired student's t-test in G. H Representative western blot showing SLN protein in soleus muscle of 8-10 week old WT and KO animals housed at RT. GAPDH was used as a loading control. I Alignment of the genomic region 1.5 Kb upstream of the mammalian Sln gene using the ECR Browser (http://ecrbrowser.dcode.org/). Human was used as the reference genome. The location of conserved NFAT binding sites are indicated by blue diamonds and CREB sites by yellow. Sites were conserved across all mammalian species available in the database except opossum. Mouse, rat, and dog are provided as representative examples. The structure of the Sln-Luc reporter construct is aligned below. J Activity of the Sln-Luc reporter in C2C12 myoblasts transiently transfected with an empty control vector or ones expressing constitutively active calcineurin (CnA*) and Rcan1. Luciferase activity was normalized to beta-galactosidase activity from a co-transfected vector. (n=3, assayed in duplicate). Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD. * = p < 0.05; ** = p < 0.01. (two-way ANOVA with multiple comparisons