Ab176328

The cells and mouse heart tissue were homogenized in lysis buffer to extract whole-cell lysates, and the protein concentrations of the samples were determined by BCA kit. The cleavage products were examined by Western blotting assay as described previously (He et al., 2015a (link)). The protein concentration of each sample was adjusted to the same level, and 10 μL of each sample was loaded into a gel for electrophoresis. The protein samples were then separated by SDS-PAGE, and the proteins in the gel were transferred electrically to a polyvinylidene fluoride (PVDF) membrane. Non-specific binding was blocked by incubating the membranes in 5% BSA. Afterwards, the PVDF membranes and antibodies were incubated at 4°C overnight, and the corresponding horseradish peroxidase secondary antibody (ZSGB-BIO, goat anti-rabbit ZB-2301) was added for 1 h. The membranes were washed, and the images were recorded and analyzed with a Bio-Rad Bole ChemiDoc MP Chemiluminescent Gel Imaging System. Antibodies against the following proteins were used: PPARγ (Abcam, ab272718, London, United Kingdom, 1:1000), PGC1α (Abcam, ab176328, London, United Kingdom, 1:1000), and β-actin (NCM Biotech, AB1020, Suzhou, Jiangsu, China, 1:1000).

Cells were grown in 24-well chamber slides (NEST, USA) for 48 h, and washed with cold PBS and fixed in 4% paraformaldehyde for 15 min. Subsequently, the coverslips were permeabilized with 0.3% Triton X-100 for 6 min and blocked with 5% normal goat serum for 30 min at room temperature. Then, the cells were incubated with anti-PGC-1β (ab176328, Abcam) and anti-FOXA2 (ab117542, Abcam) overnight at 4 °C, and followed by incubation with fluorescent secondary antibody for 1 h at room temperature. The nucleus was visualized with DAPI solution (dilution: 1:1000, Sigma, USA) for 6 min in the dark. Confocal laser scanning microscope was utilized for image analysis.

After the mice were sacrificed, tissues were immediately collected, snap-frozen in liquid nitrogen, and stored at –80°C for downstream analyses. Muscles were lysed in 200–350 μL ice-cold 1% Triton X100 in PBS containing proteinase inhibitors (Roche Diagnostic), homogenized using TissueLyser (QIAGEN), and 25 μg of protein lysate was processed as described previously (13 (link)). Primary antibodies — mouse monoclonal anti-FGF1 (AF4686, R&D Systems), mouse monoclonal anti-GAPDH (sc-59540, Santa Cruz Biotechnology Inc.), rabbit polyclonal anti–HO-1 (ADI-SPA-894, Enzo Life Sciences), rabbit polyclonal anti-AMPKA (2603S, Cell Signalling Technology), and rabbit monoclonal anti–PGC1-β (ab176328, Abcam) — and secondary antibodies (conjugated with HRP) include anti–mouse Ig (554002, BD Biosciences) for the detection of GAPDH or FGF1, as well as anti–rabbit IgG (7074, Cell Signaling Technology) for the detection of HO-1, AMPKA, PGC1-β, were used.