Chemiluminescence reagent

THP-1-derived MΦs and isolated EVs were lysed in RIPA Buffer (Thermo Fisher Scientific) supplemented with protease and phosphatase inhibitor cocktails (Roche), followed by centrifugation at 14,000 ×g for 15 min at 4°C. Protein quantities of the lysates were quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). For Western blotting, fractionation of lysates was carried in sodium dodecyl sulfate polyacrylamide gel electrophoresis on NuPAGE 4%–12% Bis-Tris Protein Gels (Thermo Fisher Scientific). Then, fractionated samples were transferred onto nitrocellulose membranes for immunoblotting. The membranes were blocked in Tris-buffered saline containing 5% powdered bovine serum albumin (BSA) for 1 h at room temperature, and then incubated overnight at 4°C with anti-TSG101 rabbit polyclonal antibody (Abcam) and anti- Calnexin rabbit polyclonal antibody (Abcam), which were diluted 1:1000. After incubation with an appropriate anti- HRP conjugate (Thermo Fisher Scientific), bands were visualized with a chemiluminescence reagent (Biological Industries) and detected using the ChemiDoc system (Bio Rad, CA).

The CEK cells were treated with PBS or different dosages of Hg for 24 h. In parallel, the CEK cells were exposed to the 15-μmol/L Hg for 24 h in the presence or absence of 1-μmol/L 4-PBA. The CEK cells were collected followed by cell lysis with lysis buffer (Beyotime Institute of Biotechnology, Wuhan, China). At 4°C, cell lysis suspension was centrifuged at 13,000 × g for 25 min. Total protein concentrations were measured using the BCA protein assay kit (Beyotime Institute of Biotechnology, Wuhan, China). Equally denatured protein samples were separated with sodium dodecyl sulfate polyacrylamide gel electrophoresis gels and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA) and blocked. The membranes were probed with specific primary antibody for 2 h at room temperature followed by adding secondary antibody. The immune-relative bands were visible with a chemiluminescence reagent (Biological Industries, Beit Haemek, Israel), and the data were analyzed by densitometry. Glyceraldehyde-3-phosphate dehydrogenase was the protein-loading control.