Nlrp3

RIPA Lysis buffer (50 mM Tris-HCl pH 7.5, 1% NP-40, 0.5 mM ethylenediaminetetraacetic acid[EDTA], 0.1% SDS, 150 mM NaCl, 0.5% sodium deoxycholate) containing PMSF and protein phosphatase inhibitors was used to extract proteins from lung tissues, and then BCA protein assay (Beyotime, China) was used to determine the protein concentration of the supernatant. The protein samples are separated on SDS-PAGE gels and then transferred to NC membranes (PALL, USA). After blocking with 5% skimmed milk in TBST at room temperature for 2h, the membrane was incubated overnight at 4°C with primary antibodies for RBM3 (1: 500), Bax (1: 5000), Bcl-2 (1: 1000) (proteintech, China), caspase-1 (1: 500), caspase-3 (1: 500), phospho-NF-κB p65 (Ser536, 1: 200), NF-κB p65 (1: 500), IκBα (1: 500) (Santa Cruz Biotechnology, CA, USA), phospho-IκBα (1: 1000), IL-1β (1: 1000) (A nity, Biosciences, China), NLRP3 (1: 500) and βactin (1: 5000) (Beyotime, China). Appropriate secondary antibodies in TBST buffer with 5% skim milk were used to incubate the membranes at room temperature for 1 h, and then immunoblotting bands were detected via BeyoECL Star chemiluminescent substrate (Beyotime, China). Finally, protein bands were analyzed with Image J software (version 1.31, USA).