A1192

Protein samples from cells and tissues were extracted and total protein concentration was determined using a BCA kit (Beyoncé, P0012, Shanghai, China). The total protein in the samples was separated by 6%–12% SDS-polyacrylamide gel electrophoresis and then transferred to 0.22 μm polyvinylidene difluoride membranes. Subsequently, the membranes were incubated with primary antibodies, followed by incubation with the corresponding secondary antibodies. The target bands were developed using the ECL chemiluminescence kit (Servicebio, Wuhan, China), and grayscale values were analyzed by ImageJ software. The following antibodies were used: HRP-labeled goat anti-rabbit (1:10,000, ZhongShan JinQiao, ZB-2301, Beijing); HRP-labeled goat anti-mouse (1:10,000, ZhongShan JinQiao, ZB-2305, Beijing); anti-GAPDH (1:50,000, proteintech, 60004-1-IG, United States); anti-HDAC2 (1:1,000, A19626, ABclonal); anti-STAT3 (1:1,000, A1192, ABclonal); anti-p-STAT3 (1:2,000, 9145T, CST); anti-GPX4 (1:1,000, ABclonal, A1933); anti-SLC7A11 (1:1,000, Proteintech, 26864-1-AP, United States); anti-ACSL4 (1:1,000, Abclonal, A6826); anti-CD9 (1:1,000, Abcam, ab92726); anti-TSG101 (1:1,000, Abcam, ab125011).

Total proteins were extracted from homogenized mouse lung tissues or HPASMCs and the protein concentration was measured using a BCA protein concentration assay kit (Beyotime Institute of Biotechnology, China). The protein samples were separated by SDS‐PAGE and transferred to PVDF membranes, followed by blocked with 5% skim milk powder solution (Inner Mongolia Yili Industrial Group Co., Ltd., China)/3% BSA (Labgic Technology Co., Ltd., China) for 1 h. The molecular weights of protein blots can be distinguished by the loading protein marker. The PVDF membranes were cut according to the protein marker prior to hybridization with primary antibodies. Then membranes were incubated overnight at 4 °C with primary antibodies against SOCS5 (A7952, 1:1,000), JAK2 (A11497, 1:1,000), p-JAK2 (AP0531, 1:1,000), STAT3 (A1192, 1:1,000), p-STAT3 (AP0705, 1:1,000, all from ABclonal Technology Co., Ltd., China), β-actin (sc-47778, 1:1,000, santa cruz biotechnology, inc., USA) and incubated with HRP (1:5,000, Beyotime Institute of Biotechnology, China) bound goat anti-rabbit IgG (A0208)/goat anti-mouse IgG (A0216) at 37 °C for 45 min. All antibodies were diluted with 5% skim milk powder solution. Finally, the protein bands were detected by the ECL system (Beyotime Institute of Biotechnology, China).