Horseradish peroxidase conjugated anti rabbit secondary antibody

Mice lung tissues were lysed with RIPA buffer (Applygen, Beijing, China). The supernatants were collected and quantified with a BCA assay kit (Solarbio, Beijing, China) Then, protein extracts from tissues were separated on a 10% sodium dodecyl sulfate–polyacrylamide gel and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, United States). After being blocked with 5% nonfat milk, the membranes were probed with specific primary antibodies against β-actin, TLR4, Syk, phospho-Syk (p-Syk) (Y525/526), PKC, p-PKC, NF-κB p65, and p-NF-κB p65 (Ser536) (all from Cell Signal Technology, United States) separately overnight at 4°C and incubated with horseradish peroxidase–conjugated anti-rabbit secondary antibody (Thermo Fisher, United States) for 2 h. The labeled protein bands were visualized using an enhanced chemiluminescent (ECL) substrate kit (YEASEN Biotech, Shanghai, China), and digital images were captured by a chemical imaging system (Clinx Science Instruments Co., Ltd, China). The density of each band was quantified by ImageJ.

Cells either treated or non-treated with drugs were harvested, washed with cold phosphate-buffered saline (PBS) and lysed in radio immunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.4, 1% NP40, 0.1%SDS, 150 mM NaCl, 5mM EDTA) containing freshly added protease and phosphatase inhibitors cocktail purchased from Thermo Scientific, USA or cells were lysed with 2X sample buffer [ 62.5 mM Tris-HCl pH (6.8), 2 % (w/v) SDS, 10 % glycerol, 50 mM dithiothreitol, 0.1 % (w/v) bromophenol blue]. Lysed cells were centrifuged at 13,000 g for 15 min, lysate was used for protein quantification using BCA protein assay kit (Thermo scientific), according to the manufacturer's instructions. Proteins were resolved by SDS-PAGE (polyacrylamide gel electrophoresis) and transferred to a nitrocellulose membrane (Whatman^R, PROTRAN^R). Membranes were blocked in 1% BSA in PBST (BSA/PBS/1% Tween) for 1 hour. Primary antibodies at a 1:1000 dilutions were added in 1% BSA in 1X PBST and incubated overnight at 4⁰ C. Membranes were washed three times with 1X PBST and then incubated with horseradish peroxidase conjugated anti-rabbit secondary antibody (Invitrogen) at a dilution of 1:5000 for 1 hour at room temperature and immunoblotting was performed using ECL detection kit reagent (BIO-RAD, Clarity™ Western ECL Substrate, 200 ml #1705060).