Heat shock protein 70 hsp70

After cell proteins were extracted, they were heated at 100 ℃ for 3 min to denature them. The protein was transferred to the negative control (NC) membrane, and the NC membrane was sealed in 5% skim milk powder [phosphate-buffered saline (PBS) preparation] at 37 ℃ for 2 h or 4 ℃ overnight. The primary antibody was then added to bind the target protein. The primary antibodies—CD9 (Abcam, Cambridge, UK), CD81 (Abcam, Cambridge, UK), Tumor susceptibility gene 101 (TSG101) (Abcam, Cambridge, UK), Heat Shock Protein 70 (HSP70) (Abcam, Cambridge, UK), ALG-2-interacting protein X (ALIX) (Abcam, Cambridge, UK), and flotillin-1 (Abcam, Cambridge, UK)—were incubated in a shaker at room temperature for 2 h or overnight at 4 ℃. The secondary antibody of horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (Abcam, Cambridge, UK) or goat anti-mouse (Abcam, Cambridge, UK) was incubated and incubated in a shaker for 1 h or overnight at 4 ℃. Diaminobenzidine (DAB) kit was used to develop color. ImageJ software (Bethesda, Maryland, USA) was used for gray value analysis of protein bands. Experimental results were recorded, and the NC film was dried, scanned, and preserved.

Total protein was extracted from CAFs-derived exosomes or supernatant using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology). Briefly, 40 mg protein were loaded on each lane, separated using 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. Following blocking with 5% skim milk at room temperature for 30 min, the membranes were subsequently incubated with the primary antibodies (all 1:1,000) against CD9 antigen (cat. no. ab92726; Abcam), CD63 antigen (cat. no. 55051; Cell Signaling Technologies, Inc.), tumor susceptibility gene 101 protein (cat. no. ab125011; Abcam), heat shock protein 70 (HSP70; cat. no. 4873; Cell Signaling Technologies, Inc.), CD81 antigen (cat. no. 10037; Cell Signaling Technologies, Inc.) overnight at 4°C, and with horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (cat. no. G-21234; Invitrogen; Thermo Fisher Scientific, Inc.; 1:5,000) at room temperature for 2 h. Blots were visu-alized using chemiluminescence detection (GE Healthcare) and analyzed by ImageJ 1.8.0 software (National Institutes of Health). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; cat. no. D190090; Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.) was applied as an internal reference.