2.6. Western blotting

Total protein was extracted from cells and tissues by using RIPA lysis buffer (Beyotime, Shanghai, China). Total protein concentration was measured by the BCA Protein Assay Kit (Beyotime, Shanghai, China). Then proteins were separated on SDS‐PAGE under the following conditions: 70 V for 30 min, followed by 120 V for 90 min. And then the protein bands were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA) at 300 mA for 2 h. After being blocked with 5% nonfat milk for 2 h at room temperature, the membranes were incubated overnight at 4°C with the following primary antibodies: HSPB1 antibody (1:500, Abcam, ab62339), CD63 antibody (1:400, Abcam, ab134045), CD81 antibody (1:400, Abcam, ab109201), TSG101 antibody (1:300, Abcam, ab30871), Alix antibody (1:300, Abcam, ab117600), GXP4 antibody (1:1000, Abcam, ab75801), FTH1 antibody (1:500, Abcam, ab75972), Nrf2 antibody (1:300, Abcam, ab137550), HO‐1 antibody (1:400, Abcam, ab68477), and POR antibody (1:500, Abcam, ab257595), Cyb5r1 antibody (1:300, Abcam, ab104217) and β‐tubulin antibody (1:800, Abcam, ab176560). Then, the membranes were incubated with horseradish peroxidase (HRP)‐conjugated goat anti‐rabbit IgG (1:2000, Abcam, ab6721) at 37°C for 1 h. The protein bands were visualized by using the Enhanced chemiluminescence reagents (Millipore, MA, USA) in a Gel Imaging System (Thermo Fisher Scientific). The expression of the relative protein was evaluated by the grey value ratio of the target protein to the internal reference GAPDH and analysed with ImageJ software (Thermo Fisher Scientific).