Enhanced chemiluminescent agent

Western blot analysis was performed as previously described (11 (link)). In brief, the diaphragms were rinsed with ice-cold PBS, harvested with radioimmunoprecipitation assay buffer (Applygen, Beijing, China) and quantified using a bicinchoninic acid protein assay (WellBiology, Changsha, China). Total protein (30 µg/lane) was separated by 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked in 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 1 h at room temperature and probed with antibody against myostatin (cat no. 19142-1-AP) and β-actin (cat no. 20536-1-AP; 1:1,000; Proteintech, Chicago, IL, USA) at 4°C overnight, followed by incubation with a secondary antibody (cat no. SA00001-2; 1:5,000; Proteintech, Chicago, IL, USA) conjugated to horseradish peroxidase at room temperature for 1 h. Immunoreactivity was detected by enhanced chemiluminescent agent (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions. The protein expression levels were quantitatively analyzed and normalized against the β-actin loading control.

Proteins harvested from the cells and stomach muscularis tissues were homogenized in lysis buffer (radioimmunoprecipitation assay: phenylmethyl sulfonyl fluoride = 100:1). Protein concentration was evaluated using the bicinchoninic acid method. Equal amounts of protein were subjected to 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. After soaking in 10% nonfat dry milk at room temperature for 1 hour, membranes were incubated with specific primary antibodies (Table 1) at 4°C overnight. Then the membranes were incubated with specific secondary antibodies (AntGene, Wuhan, China) for 1 hour at 37°C. Finally, the bands were visualized by chemiluminescence immunoassay using an enhanced chemiluminescent agent (Thermo Fisher Scientific Inc, Waltham, MA). The intensities of the bands were quantified using ImageJ v1.51.