Antibodies for β catenin

Whole protein lysates from all tissues were prepared as previously described.^{41 (link)} Whole protein lysates from cell lines were prepared by using RIPA lysis buffer (Santa Cruz Biotechnology Inc., Dallas, Texas, USA) according to the manufacturer’s instructions. Nuclear proteins were purified using a nuclear extraction kit (EMD Millipore, Billerica, MA, USA). SDS-polyacrylamide gel electrophoresis and western blot analyses were performed as previously described.^{33 (link)} Antibodies for nuclear factor of activated T cells c1 (1:1 000) and lamin B1 (1:1 000) were from Santa Cruz Biotechnology. Antibody to detect irisin (epitope 42–112, 1:500) and FNDC5 expression was purchased from Phoenix Pharmaceuticals Inc. (#067-17). Antibodies for β-catenin (1:10 000) were from Sigma and those from P-AKT-1 (1:1 000), calcineurin (1:1 000), P-JNK (1:1 000), and β-actin (1:1 000) were from Cell Signaling (Danvers, MA, USA). The secondary antibodies were horseradish peroxidase-linked goat-anti-rabbit IgG (Santa Cruz Biotechnology). Blots were visualized using Pierce ECL chemiluminescence kit (Thermo Fisher Scientific).

Western blotting was carried out as described previously [32 (link)]. The antibodies for HMGCS2 and PPARγ were obtained from Abcam (Cambridge, MA, USA). The antibodies for β-catenin and β-actin were purchased from Sigma-Aldrich. The antibodies for Axin2 and c-Myc were acquired from Cell Signaling (Danvers, MA, USA). The anti-PPARα antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Densitometric quantification from three separate experiments was done using ImageJ.