Total protein

Cells were lysed with RIPA containing protease inhibitors to obtain the total protein (Thermo Fisher Scientific). The protein was separated by 10% SDS-PAGE and then transferred into PVDF membrane. The membrane was incubated with the primary antibody GOLM1 (1:1000, Sigma Aldrich, US) at the temperature of 4°C overnight. Then, membrane was incubated with secondary antibody (1:5000, Sigma Aldrich, US) under room temperature for 40 min. The bands were visualized with ECL detection reagents (Amersham Biosciences, Sweden).

Lung tissues or MLE-15 cells were lysed with RIPA containing protease inhibitors to obtain the total protein (Thermo Fisher Scientific). The protein was separated by 10% SDS-PAGE and then transferred to a PVDF membrane. The primary antibodies against JNK (1:1000, #9252; Cell Signaling Technology, Danvers, MA,USA), p-JNK (1:1000, #9255; Cell Signaling Technology), Bcl-2 (1:1000, SAB4500003; Sigma Aldrich), Bax (1:1000, B8429; Sigma Aldrich), and Bak (1:1000, SAB4502532; Sigma Aldrich) were incubated with the membrane at 4℃ overnight. Thereafter, the membranes were incubated with secondary antibody (1:5000, Sigma Aldrich) at room temperature for 40 min. The bands were visualized with ECL detection reagents (GE Healthcare, Little Chalfont, Buckinghamshire, UK). GAPDH served as the internal reference, and the ratio of the gray value of target band to internal reference band was considered as the relative protein level. ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to analyze the gray value.

Total protein (Thermo Fisher Scientific, Waltham, MA, USA) and lactate dehydrogenase (LDH) cytotoxicity (Thermo Fisher Scientific) were determined in BALF. α₁-antitrypsin (Cusabio, Wuhan, Hubei, China), neutrophil elastase (Cusabio), 8-isoprostane (Cayman, Ann Arbor, MI, USA), interleukin (IL)-6 (R&D Systems, Minneapolis, MN, USA), caspase-3 (Elabscience, Houston, TX, USA), ACE2 (Elabscience), AngII receptor type 1 (AT1) (Elabscience) and AngII receptor type 2 (AT2) (Elabscience) were determined in lung lysates using ELISA. All analyses were in accordance with the manufacturers’ instructions. Data obtained from lung lysates are presented after adjusting for the β-actin level (Cell Signaling, Danvers, MA, USA).