Ecl plus western blotting detection reagent

The NRCMs or cardiac tissues were lysed using RIPA buffer (Beyotime, Nantong, China) with 1 mM PMSF (ST505, Beyondtime. Nantong, China), and the concentration of protein samples was evaluated by BCA protein assay kit (Beyotime, Nantong, China). Equal amounts of protein were separated in 10% SDS-PAGE and transferred into PVDF membranes which incubated with 5% milk and with primary antibody at 4 °C overnight. The primary antibodies were used as follow: PPARγ (1:1,000, Proteintech Group, 16643-1-AP, Wuhan, China), PGC-1α (1:1,000, Novus Biologicals, NBP2-37562, Littleton, COLO, USA), Collagen I (1:1,000, Wanleibio, WL0088, Shenyang, China), α-SMA (1:4,000, Proteintech Group, 23081-1-AP, Wuhan, China), GAPDH (1:1,000, Kangchen, KC-5G4, Shanghai, China). The next day, the membranes were incubated with the secondary antibodies and signals were visualized using the ECL Plus Western blotting detection reagents (Bio-Rad, Hercules, CA, USA) and the ChemiDoc XRS Plus luminescent image analyzer (Bio-Rad, Hercules, CA, USA). Densitometric analysis of band intensity was performed using Imagelab software (Bio-Rad, Hercules, CA, USA). GAPDH was used as loading controls for total protein expression.

Western blotting was performed as previously described [48] (link). Briefly, using RIPA lysis buffer (Thermo Fisher Scientific, USA) containing protease and phosphatase inhibitors (Thermo Fisher Scientific, USA) was used collect all proteins. The protein concentrations in the extracts were detected using the BCA assay (Thermo Fisher Scientific, USA). Equal amounts of the protein sample were loaded with sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE). Then, the protein sample was electrotransferred to 0.22 μm polyvinylidene difluoride membranes (PVDF). The membranes were blocked with 5% BSA dissolved in TBST for 2 h at room temperature and incubated with the indicated primary antibodies: anti-MMP2, anti-MMP9, anti-PI3K, anti-p PI3K, anti-AKT, anti-p AKT, and anti-β-actin overnight at 4 °C, followed by incubation with the appropriate secondary antibody for 2 h at room temperature. After washing with TBST buffer, protein bands were visualized using ECL plus Western blotting detection reagents (Bio-Rad, USA). The strips were scanned and quantified using a computer image analysis system. Information on the antibodies used in the Western blot is listed in Table S5. All results are representative of three independent experiments.

Immunoblots were carried out using protein lysates obtained from undifferentiated pPICs and pPICs that had undergone early myogenic differentiation (n = 3/group). Approximately, 50 μg of protein were separated on gradient (10% to 15%) SDS-polyacrylamide gels. After electrophoresis, proteins were transferred onto nitrocellulose membranes and blocked with 5% milk, then incubated with antibodies against IGF-1 (Santa Cruz Biotechnology, Dallas, Texas), HGF (Santa Cruz Biotechnology), transforming growth factor (TGF)-β1 (Santa Cruz Biotechnology), neuregulin (NRG)-1 (Santa Cruz Biotechnology) at dilutions suggested by the manufacturers. GAPDH (Millipore) was used as a loading control. Proteins were detected by chemiluminescence using horseradish peroxidase–conjugated secondary antibodies (Santa Cruz Biotechnology) and visualized using ECL Plus Western Blotting Detection Reagents (Amersham, Little Chalfont, United Kingdom) and a Chemidoc imaging system (Bio-Rad).