Af5234

The lung tissues or PASMCs were homogenized in RIPA lysis buffer (Beyotime, Shanghai, China) with a protease and phosphatase inhibitor cocktail (Beyotime, Shanghai, China). Samples containing 20–40 μg of protein were separated by 10% SDS-PAGE gel and then transferred to polyvinylidene fluoride (PVDF) membranes (G.E. Healthcare, Germany). After blocking with 5% skim milk or 5% BSA, the PVDF membranes were incubated overnight on a shaker at 4°C with the following primary antibodies: collagen1 (AF6524, 1:1,000, Beyotime), collagen3 (AF6531, 1:1,000, Beyotime), MMP2 (AF0234, 1:1,000, Beyotime), MMP9(AF5234, 1:1,000, Beyotime), PCNA (AF0261, 1:1,000, Beyotime), SRC (AF1831, 1:1,000, Beyotime), p-SRC (AF5923, 1:1,000, Beyotime), PIM1 (AF1807, 1:1,000, Beyotime), eNOS (AF6792, 1:1,000, Beyotime), and α-tubulin (sc-5286, 1:500, Santa Cruz). After incubation with horseradish peroxidase (HRP)-linked secondary antibody (Beyotime, Shanghai, China), the protein bands were imaged through Molecular Imager ChemiDoc XRS System (Bio-Rad, Philadelphia, USA). Densitometric quantification was performed by ImageJ (NIH, USA). The α-tubulin served as a loading control.

Western blot analysis was performed to detect protein expression levels. Proteins were extracted from cell lysates using a cell lysis buffer (Beyotime, Shanghai, China) and separated by 10% SDS-PAGE followed by electrotransfer onto nitrocellulose membranes, blocking with skimmed milk at room temperature for 1 h. Membranes were incubated overnight at 4°C with primary antibodies, including anti-Nectin2 (ab135246; Abcam, Cambridge, USA), anti-GAPDH (AF1186; Beyotime), anti-β-actin (ab8226; Abcam), anti-MMP2 (AF1420; Beyotime), anti-MMP9 (AF5234; Beyotime), anti-cleaved caspase-3 (ab32042; Abcam), anti-bax (bs-0127R; Bioss, Beijing, China), anti-bcl2 (AF6285; Beyotime), anti-N-cadherin (AF0243; Beyotime), anti-E-cadherin (AF1552; Beyotime) and anti-ANXA2 (AF5115; Beyotime). Membranes were washed several times with TBST and incubated with horseradish peroxidase-conjugated secondary antibodies (KC5G5; Aksomics, Shanghai, China) at room temperature for 1 h. Protein blots were visualized using an ECL kit (P0018FS; Beyotime), and protein levels are expressed as the ratio of band optical intensity relative to that of GAPDH. Densitometry analyses were performed using ImageJ software (version 1.52; NIH, Bethesda, USA).