Protease and phosphatase inhibitors

The HUVECs were seeded in a 6-well plate with a density of 2 × 10⁵ cells per well. These cells were then treated with 1 μg/ml rhVEGF₁₆₅ and combined with the previously indicated treatments. After 24 h of incubation, the cells were rinsed three times with PBS. The cell lysates were harvested with a lysis buffer with protease and phosphatase inhibitors (Sangon Biotech, China). After lysing on ice for 30 min, the cell lysates were centrifuged at 14,000 × g for 15 min at 4°C. The protein concentration was calculated using a bicinchoninic acid protein assay kit (Thermo Scientific, United States) and normalized. Thirty micrograms of protein were resolved with 6–10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electro-transferred to polyvinylidene fluoride membranes. The membranes were then blocked with a protein-free rapid blocking buffer (Epizyme, China) and immunoblotted with the following antibodies: rabbit monoclonal anti–β-catenin (Cell Signaling Technology, United States, 1:1000), rabbit monoclonal anti-Axin 1 (Cell Signaling Technology, United States, 1:1000), and rabbit monoclonal anti–β-actin (Cell Signaling Technology, United States, 1:1000). All quantitative data were normalized to β-actin as an endogenous control. ImageJ software was used to analyze the band intensity.

Mechanically homogenised frozen hearts and fresh cell suspensions were lysed in RIPA buffer (Beyotime, China) with protease and phosphatase inhibitors (Sangon Biotech) on ice for 30 min. Protein concentrations were estimated using BCA assay (Beyotime) and subsequently adjusted to equal levels. Samples (30 μg) were loaded onto polyacrylamide gels and hybridised onto polyvinylidene difluoride membranes (Millipore, USA). Membranes were blocked with 5% BSA for 1 h and probed with the relevant primary antibodies for 16 h at 4°C. After washing, the membranes were incubated at room temperature for 1 h with appropriate secondary antibodies (CST, USA). The blots were developed using ECL (Millipore). The primary antibodies were GAPDH (CST, 5174), Cypher (Abnova, H00011155-M06), β-catenin (Abcam, 32572), β-catenin Ser675 (CST, 4176), β-catenin Ser552 (CST, 9566), β-catenin Ser33/37/Thr41 (CST, 9561), Gsk-3β (CST, 12456), Gsk-3β Ser9 (CST, 5558), vimentin (Abcam, 92547), vimentin Ser72 (Abcam, 52944), stathmin (Abcam, 52630), stathmin Ser16 (Abcam, 47382), troponin I (Abcam, 209809), troponin I Ser23+24 (Abcam, 190697), cyclin D1 (Abcam, 134175), c-Myc (Abcam, 32072), c-jun (Abcam, 32137), Myc (CST, 2276), Flag (Beyotime, AF519), and rabbit IgG (CST, 2729). The ImageJ software was used to quantify the relative protein levels.