Vascular endothelial growth factor (vegf)

Lung tissues and PASMCs were lysed in radio immunoprecipitation assay (RIPA) lysis buffer (containing 1% PMSF) on ice to extract total proteins. Nuclear and cytoplasmic proteins were extracted following the manufacturer's recommendations of the nuclear and cytoplasmic extraction kit (Beyotime). After protein quantification, the same amount of protein from each sample were separated on sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE), and then the proteins on the gel were transferred to PVDF membranes (Millipore) by semi‐dry electrophoretic transfer system (Bio‐rad). The membranes were then blocked with 5% BSA and incubated with primary antibodies against calpain‐1 (1:1000, proteintech), HIF‐1α (1:1500, ABclonal), P65 (1:2000, proteintech), β‐actin (1:5000, proteintech), Lamin B (1:10000, proteintech), VEGF (1:1000, ABclonal), TGF‐β1 (1:800, Wanleibio), PCNA (1:1500, ABclonal), MMP2 (1:1000, ABclonal) and collagen I (1:1000, Wanleibio) overnight at 4°C. On the second day, membranes were incubated with HRP‐conjugated secondary antibodies (1:10000, ABclonal). The immune response bands were visualized with a chemiluminescence reagents (Biosharp).

IP lysis buffer with a protease inhibitor cocktail was used to harvest the protein from the cells. 40 μg of total protein was transferred onto a PVDF membrane. The membrane was subjected to immunoblotting by incubating with specific primary antibodies overnight at 4°C. Antibody signals were detected by ChemiDox XRS+.
For immunoprecipitation experiments, cells were harvested, lysed, quantified, and precleared with 20 μL of Dynabeads™ Protein G (ThermoFisher Scientific, #10004D, USA). After centrifugation, the supernatant was collected and incubated with antibody at 4°C overnight. Dynabeads™ Protein G was added to the protein‐antibody complex for additional incubation. Subsequently, the beads were collected and boiled. Western blot analysis was performed on the samples, with 20 μg of total protein used as the input control.
The antibodies for Western Blot: HIF‐1α (CST, #36169S, 1:1000), Hydroxylation HIF‐1α (CST, #3434T, 1:1000), PHD3 (Novus, #NB100‐139, 1:1000), GLUT1 (CST, #12939S, 1:1000), RP58 (Proteintech, #12714‐1‐AP, 1:1000), PHD2 (CST, #4835S, 1:1000), VEGF (Abclonal, #A12303, 1:500), N‐cadherin (CST, #13116, 1:1000), HK2 (Proteintech, #22029‐1‐AP, 1:2000), Ubiquitin (CST, #3936S, 1:1000), PGK1 (Proteintech, #17811‐1‐AP, 1:1000), β‐actin (Sigma, #A5441, 1:10000), ABCG2 (Sigma‐Aldrich, MAB4146, 1:1000), and anti‐Flag (CST, #14793, 1:1000).