Low melting point agarose gel

Mouse flow cytometry antibodies CD16/CD32 (553141), CD45 APC-Cy7 (557659), CD11b PE (553311), Ly-6G and Ly-6C FITC (553127), and flow cytometry buffer (Stain Buffer FBS) were purchased from BD Pharmingen (United States). The red blood cell lysate was purchased from Biyuntian Company (China). Type II lung epithelial cell isolation and identification antibody Biotinylated anti-mouse CD45, Biotinylated anti-mouse TER119, Anti-mouse EpCAM-APC were purchased from eBioscience (United States). Biotinylated anti-mouse Integrin β4, Biotinylated anti-mouse CD31 were purchased from Biolegend (United States). Streptavidin-PE, Biotinylated anti-mouse CD16/32 were purchased from BD Pharmingen (United States). Dynabeads^® MyOne^TM streptavidin T1 magnetic beads, Rabbit anti-pro-SP-C, and DAPI were purchased from Thermo Fisher Scientific (United States). Dispase II, low melting point agarose gel, DNase I were purchased from Sigma (United States). VWF, VWFpp polyclonal antibody and vimentin polyclonal antibody were purchased from Proteintech (United States). Mcp1 Monoclonal antibody was purchased from Proteintech (United States). Mouse antibodies against CD63, Tsg101, and CD81 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States). Rabbit antibodies against PTEN, Phospho-NF-κB p65 Phospho-Akt, Calnexin were purchased from Cell Signaling Technology (MA, United States).

Mutants of the PNPase gene for in vitro studies were prepared by two successive PCR reactions. In the first step, two fragments were PCR-amplified using the wild-type PNPase gene as a template: one using the forward primer PNPaseNcoFor and a reverse primer introducing the mutation (primers PNPaseS1x2Rev or PNPaseKHx2Rev or PNPaseS1x4Rev) (Table S1); the other using a reverse primer PNPaseNotRev and a forward primer introducing the mutation (primers PNPaseS1x2For or PNPaseKHx2For or PNPaseS1x4For) (Table S1). PCR products were resolved on 1% low melting point agarose gel (Sigma), the bands of interest were excised and after melting the matrix at 70°C, they were mixed in one PCR reaction which amplified the entire PNPase gene, with mutations, using primers PNPaseNcoFor and PNPaseNotRev. The product of the last PCR was digested with NcoI and NotI (NEB), resolved on a low melting point agarose gel, and the gel band was directly ligated with T4 ligase (NEB) into a pET duet plasmid, which had been digested with the same restriction enzymes and dephosphorylated with CIP (NEB) according to the manufacturer’s instructions.