Horseradish peroxidase linked goat anti rabbit igg

Cells were washed with phosphate-buffered saline (PBS) and homogenized in cell lysis buffer (150 mM NaCl, 1% NP40, 10% DOC, 10% SDS, 50 mM Tris, pH 7.4). Determination of protein concentration was performed using the BCA protein assay kit (Pierce). Samples were mixed with standard Laemmli loading buffer and 30 μg protein loaded on mini-Protean TGX precast gel (Bio-Rad). After SDS-PAGE, proteins were transferred to Immunoblot PVDF membranes (BioRad), the membrane blocked with 3% BSA, and further incubated with rabbit monoclonal anti-DIMT1 antibody (1:1000 dilution) and Rabbit polyclonal anti-OXPHOS (1:250 dilution antibody; both from Abcam), anti-NBR1 and anti-DNAJC19 (1:2000 dilution antibody; both from Sigma Aldrich), anti-NOB1 and anti-PES1 (1:1000 dilution; Sigma Aldrich) Tubulin and β-actin antibody (1:5000 dilution; Abcam) was used as a loading control. Horseradish-peroxidase-linked goat anti-rabbit IgG (1:5000 dilution; Santa-Cruz Biotechnology) was used as a secondary antibody. Blots were developed with enhanced chemiluminescence (ECL). Densitometry analysis was performed using BioRad ImageLab software.

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).