Primary antibody against stat3

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).

Briefly, VSMCs were washed by PBS twice followed by the addition of cell lysis buffer (ABGENT, Suzhou, Jiangsu, China). Then, collection of VSMCs lysates was performed and a BCA assay kit (MaiRuiBo, Chaoyang, Beijing, China) confirmed the concentrations of proteins. SDS-PAGE (10%) was for separating VSMCs lysates, and consequent proteins were transferred on PVDF membranes, followed by 5% BSA (in TBST buffer) blockade for 1 h. Afterwards, primary antibody against STAT3 (CST, Pudong, Shanghai, China), TULP3 (abcam) or GAPDH (Protein Tech Group, Wuhan, Hubei, China) was used for probing the membranes for 12 h at 4°C. Sequentially, matched secondary antibodies were employed for incubation. A Super ECL assay kit (YRBIO, Changsha, Hunan, China) was for protein band visualization.

Proteins were extracted from the whole cell lysates and separated using 12% SDS-PAGE. After transferring to PVDF membranes (Millipore, Billerica, MA, USA), the membranes were incubated with the primary antibody against STAT3 (1:1000; Abcam, Cambridge, USA) and the internal control anti-β-actin (1:2000; Cell Signaling Technology). The membranes were incubated with secondary antibodies (1:5000; Abcam, Cambridge, USA). All bands were detected with the enhanced chemiluminescence (ECL) kit (Thermo Fisher Scientific) and quantified using ImageJ software.