Horseradish peroxidase conjugated polyclonal goat anti rabbit antibody

Protein samples (n = 3 per group, 15 μg per sample) were separated on a 5% stacking gel (62.5 mM Tris-HCl pH 6.8, 5% acrylamide/bis-acrylamide (37.5:1), 0.1% SDS, 0.125% APS, 0.055% TEMED) and a 12% separation gel (0.375 M Tris-HCl pH 8.8, 12% acrylamide/bisacrylamide (37.5:1), 0.1% SDS, 0.05% APS, 0.05% TEMED) with a mini-Protean Tetra cell (BioRad). Separated proteins were transferred onto PVDF membrane (0.45 μm, IPVH00010, Millipore) for 30 min at 1.0 A/25 V. Equal loading was assessed by Ponceau S staining. The blots were blocked (5% non-fat dry milk in Tris-buffered saline with 0.1% Tween 20) for 1 h and probed overnight at 4 °C with the primary antibodies listed in Supplementary Table 7. Detection was performed with horseradish peroxidase-conjugated polyclonal goat anti-rabbit antibody (1:2000, no. 7074, Cell Signaling; 1 h at RT), enhanced chemiluminescence substrate (32106, Pierce/Thermo Scientific) and Amersham Hyperfilm ECL films (28906837, GE Healthcare). Western blot bands were quantitatively analyzed by ImageJ 1.50i81 (link). Total protein normalization was performed on densitometric measurements of Ponceau S staining along the complete separation range of each lane. Unpaired t-test was calculated in Excel.

Protein samples were separated on 5% stacking/12% separation polyacrylamide gels with a Mini-Protean Tetra Cell (BioRad). Separated proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (0.45 μm, IPVH00010, Millipore) for 30 min at 1.0 A/25 V. Equal loading was assessed by Ponceau S staining. Blots were blocked (5% non-fat dry milk in Tris-buffered saline with 0.1% Tween 20) for 1 h and incubated overnight at 4 °C with the primary antibody (PGC-1α antibody ab54481, GAPDH antibody #2118, Cell Signaling). Detection was performed with horseradish peroxidase-conjugated polyclonal goat anti-rabbit antibody (1:2500, no. 7074, Cell Signaling; for 1 h at RT) and SuperSignal™ West Dura chemiluminescence substrate (Thermo Scientific). Fluorescence imaging was performed using Intas ECL Chemostar. Western blot band intensities were quantitatively analyzed by Adobe Photoshop CS6. Normalized signal intensities of GHR-KO and control samples were compared using the Mann–Whitney U-test.