Ab180747

The Mixed RIPA buffer (R0010, Solarbio, Beijing, China) with phenylmethanesulfonyl fluoride (PMSF) (P0100, Solarbio) at 100:1 was used to lysis cells and exosomes. The extracted proteins were quantified using the BCA Protein Assay Kit (C503021, Sangon Biotech, Shanghai, China). The antibodies used in Western blot includes rabbit anti-human CD9 (ab263019, 1:1,000, Abcam), CD63 (ab134045, 1:2,000, Abcam), HSP70 (ab181606, 1:1,000, Abcam), TSG101 (ab125011, 1:2,000, Abcam), IRAK-1 (ab180747, 1:1,000, Abcam), TRAF-6 (ab33915, 1:2,000, Abcam), GAPDH (D110016, 1:5,000, Sangon Biotech), and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (D110058, Sangon Biotech). The relative expressions of the target proteins were normalized to GAPDH.

The three groups of cells were lysed with RIPA lysis buffer on ice at 48 h after transfection. The supernatant was retained after high-speed centrifugation (12,000 × g at 4°C) and the protein concentrations were measured using a BCA kit (Beyotime Institute of Biotechnology). Equal amounts of protein (15 µg)were separated by SDS-PAGE on 10% gels (Beijing Solarbio Science & Technology Co., Ltd.), and transferred to PVDF membranes. The PVDF membranes were blocked with 5% skim milk at room temperature for 2 h. They were then incubated with primary antibodies against interleukin-1 receptor-associated kinase 1 (IRAK1; dilution, 1:1,000; cat. no. ab180747; Abcam), TNF receptor associated factor 6 (TRAF6; dilution, 1:1,000; cat. no. ab33915; Abcam) and GAPDH (dilution, 1:10,000; cat. no. ab181602; Abcam) at 4°C overnight. The membranes were then incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (dilution, 1:5,000; cat. no. BB-2202; BestBio Company; http://bestbio.bioon.com.cn/) at room temperature for 2 h. Proteins were visualized by chemiluminescence using an ECL kit (Beyotime Institute of Biotechnology), according to the manufacturer's protocols. The expression of proteins was quantified using densitometry analysis (ChemiDoc™ XRS+ gel imaging system) and analysed using image lab^™ software (version 3.0; both Bio-Rad Laboratories, Inc.).