Western luminescent detection kit

The VE-cadherin and pY685 VE-cadherin were detected using a standard Western blot protocol to determine the VE-cadherin level and the phosphorylation of Y685. The transferred membrane was blocked with 10% skimmed milk for 1 h at room temperature, and then the blocked membrane was incubated with the primary antibody against VE-cadherin (dilution 1 : 1000; category number PA1-84328, Thermo Fisher), pY685VE-cadherin (dilution 1 : 700; category number AB119785, Abcam), and β-actin (dilution 1 : 700; Santa Cruz Biotechnology) overnight at 4°C, respectively. After incubation with the horseradish peroxidase-conjugated secondary antibody (dilutions of 1 : 5000; Beijing Zhongshan-Golden Bridge Biological Technology Company, Beijing, China) for 1 h at room temperature, the immunoblotting signals were visualized using a Western Luminescent Detection Kit (Vigorous Biotechnology, Beijing, China).

The total protein was extracted from the mouse lung tissues and cultured PMVECs. Protein content was evaluated using the BCA commercial kit (Beyotime Biotechnology, Jiangsu, China). The transferred membrane was blocked with 10% skimmed milk for 1 h at room temperature, and then the blocked membrane was incubated with the primary antibody against STAT3 (1 : 1000; Abcam) and β-actin (1 : 500; Santa Cruz Biotechnology) overnight at 4°C, respectively. After incubation with the horseradish peroxidase-conjugated secondary antibody (1 : 5000; Zhong Shan-Golden Bridge Biological Technology Company, Peking, China) for 1 h at room temperature, the immunoblotting signals were visualized using a Western luminescent detection kit (Vigorous Biotechnology, Beijing, China).

Total protein was extracted from the lung tissues in mice, and the total protein and nucleoprotein were extracted from the PMVECs. The resultant protein concentrations were determined by BCA Protein Assay reagents (Beyotime Biotechnology, Jiangsu, China) according to the previous description^{18 (link)}. The expression of STAT3, pY705-STAT3, and Bcl-2 was determined using a standard protocol. The transferred membrane was blocked with 10% skimmed milk for 1 h at room temperature, and then incubated with the primary antibodies against STAT3 (1:1000; Abcam), and pY705-STAT3 (1:700; Abcam), Bcl-2 (1:700; Santa Cruz), and β-actin (1:500; Santa Cruz) overnight at 4 °C, respectively. After incubating with the horseradish peroxidase-conjugated secondary antibodies (1:5000; Zhong Shan-Golden Bridge Biological Technology Company, Beijing, China) for 1 h at room temperature, the immunoblotting signals were visualized using a Western Luminescent Detection kit (Vigorous Biotechnology, Beijing, China).