Sting 19851 1 ap

Proteins were extracted in a radioimmunoprecipitation assay (RIPA) lysis buffer, mixed with a 4 × loading buffer in a 3:1 ratio, and boiled at 100 °C for 5–10 min. The protein samples were separated on 10% SDS-PAGE gels, and then transferred to PVDF membranes. After blocking with 5% skim milk solution at room temperature (RT) for 60 min, membranes were incubated with individual primary antibodies at 4 °C overnight. The primary antibodies include anti-GFP (HT801-01, TransGen, Beijing, China), STING (19851-1-AP, ProteinTech, Wuhan, China), cGAS (sc-515777, Santa Cruz Biotechnology, Dallas, Texas, USA), IRF3 (11904S, CST, Boston, MA, USA), p-IRF3 (Ser396) (MA5-14947, ThermoFisher Scientific, Shanghai, China), TBK1 (3504S, CST, Boston, MA, USA), p-TBK1 (5483S, CST, Boston, MA, USA) and β-actin (5057, CST, Boston, MA, USA). Secondary antibody HRP-conjugated goat anti-mouse or rabbit IgG (TransGen Biotech, Beijing, China) was used to incubate the membranes for 60 min at RT. Signals were detected using enhanced chemiluminescence (ECL) substrate (Tanon, Shanghai, China), and images were visualized with an imaging system (Tanon, Shanghai, China).

Lung tissues were fixed in 4% paraformaldehyde and embedded in paraffin. For histology, lung sections (5 μm) were stained with hematoxylin and eosin (H&E). For immunohistochemistry, sections were stained with STING (19851-1-AP, Proteintech, Wuhan, Hubei, China) and Ly-6G/Ly-6c (BioLegend, San Diego, CA, USA) antibodies. For immunofluorescence, cells were stained with p-MLKL primary antibody (ab196436, Abcam, Cambridge, MA, USA), and CoraLite594-conjugated secondary antibody (SA00013-4, Proteintech, Wuhan, Hubei, China). Subsequently, pulmonary cell death was analyzed by TUNEL staining using a commercial kit (KGA702, KeyGEN BioTECH, Beijing, China). DAPI (1 µg/mL) was used to stain nuclei.