Beclin1

Antigen retrieval was performed on paraffin sections (3–5 μm) by boiling in citrate antigen retrieval buffer (pH 6.0) or EDTA buffer (pH 8.0), followed by blocking in 3% H₂O₂ for 15 min. The sections were then incubated with primary antibodies against LC3B (Abmart, Shanghai, China), Beclin1 (Abmart), p62 (Wanleibio, Shenyang, China), nuclear factor kappa B (NF-κB) p65 (Abmart), or NLRP3 (Wanleibio) at 37°C for 1 h and subsequently incubated with secondary antibodies (PV-9001, ZSGB-BIO, Beijing, China) according to the manufacturer's instructions. The expression levels of related proteins were scored semiquantitatively in a blind manner as the product of intensity scores (0, negative; 1, weak; 2, moderate; and 3, strong) and diffusion scores (1, 0-25% positive cells; 2, 26-50% positive cells; 3, 51-75% positive cells; and 4, >75% positive cells) [19 (link)].

GC cells were lysed in RIPA lysis buffer containing 50 mM Tris (pH 7.4), and the extracted protein was quantified using a BCA Protein Assay Reagent Kit (Beyotime, Shanghai, China). Each sample was then separated by electrophoresis on a 10% SDS-polyacrylamide gel and electroblotted onto a nitrocellulose membrane. The membranes were blocked with 5% skimmed milk in Tris Buffered Saline Tween (TBST) for 2 h and incubated overnight at 4 °C with primary antibodies against MLCK (Santa Cruz Biotechnology, Cat# sc-365352), phosphorylated myosin light chain (pMLC) (Cell Signaling Technology, Cat# 3674), MLC (Cell Signaling Technology, Cat# 8505), p62 (Abmart, Cat# sc-48402), Beclin-1 (Abmart, Cat# T55092), LC3B (Cell Signaling Technology, Cat# 3868) or GAPDH (Santa Cruz Biotechnology, Cat# sc-365062). The PVDF membranes were then washed three times with TBST and incubated with secondary antibodies diluted in 3% milk for 2 h at room temperature. The membranes were washed three times with TBST, and the protein bands were visualized using chemiluminescence.

Total protein was extracted from the right lung tissues using RIPA buffer (Meilunbio, Dalian, China). Proteins were separated by 10% or 12% SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, USA), which was blocked with 5% fat-free milk in TBST solution for 1 h at room temperature. The membranes were incubated with primary antibodies against LC3B (Abmart), Beclin1 (Abmart), p62 (Abmart), NF-κB p65 (Abmart), NLRP3 (Wanleibio), ASC (Cell Signaling Technology, Inc. MA, USA), pro-caspase1 (Abcam, Cambridge, UK), caspase1 p20 (Wanleibio), and mature-IL-1β (Wanleibio) at 4°C overnight. The membranes were washed with TBST and incubated with a secondary antibody (SA5-35571, Thermo Fisher, USA) at room temperature for 70 min. Immunoreactive images were detected using the Odyssey system (LI-COR Biosciences, USA) and analyzed using the ImageJ software.