Tgf beta1

The total proteins of SW480 and NCM460 cells were prepared using RIPA buffer containing protease and phosphatase inhibitors. A BCA protein assay kit was used to measure protein concentrations. 20 µg proteins were loaded per lane, separated by electrophoresis, and then transferred to polyvinylidene fluoride (PVDF) membranes (C3117, Millipore). The membrane was blocked and then incubated for 1 h with β-actin (1:100000; ABclonal, AC026), PPAR gamma (1:5000; Proteintech, 16643-1-AP), MMP9 (1:1000; Proteintech, 10375-2-AP), TNF alpha (1:3000; Proteintech, 60291-1-IG), TGF beta1 (1:2000; Proteintech, 21898-1-AP), COX2 (1: 500; Proteintech, 27308-1-AP), HIF1A (1:2500; Proteintech, 20960-1-AP) and Beta-catenin (1:10000; Proteintech, 51067-2-AP). Immunoblot analysis was performed with horseradish peroxidase (HRP)-conjugated anti-mouse antibodies or anti-rabbit antibodies (1:5000; ZSGB-BIO, ZB-5301, and ZB-5305) and developed with the ECL kit (Beyotime Biotechnology, P0018FM). The level of β-actin was used as a loading control, and the ratios of the gray value of the target protein bands to the gray value of the corresponding internal control bands were defined as the expression level of the target protein.

Total proteins were extracted from lung tissues or cells by using an ice-cold RIPA lysis buffer (Thermo) that contains protease inhibitors (Thermo). Protein concentration was determined by a BCA Protein Assay Kit (Thermo). Equal amounts of proteins were divided by SDS–PAGE and blotted to nitrocellulose membrane, followed by block with 5% skim milk. The blots were then hatched with primary antibodies against TGF-beta1 (Proteintech, China), smad3 (Abcam, USA), TET2 (Abcam, USA), GPX4 (Abcam, USA), SLC7A11 (Abcam, USA), and tubulin (Abcam, USA) at 4°C overnight. Proteins were then visualized with corresponding horseradish peroxidase- (HRP-) conjugated secondary antibodies and ECL reagents (Millipore, USA) on a gel image system. The intensity of protein bands was quantified by Image J software.