Gtx70255

For immunoblotting, primary antibodies against mCherry (rabbit; Abcam ab167453; 1:1,000), CD9 (rabbit; Abcam ab92726; 1:1,000), β‐actin (rabbit; Abcam ab8227; 1:2000), and TSG101 (mouse, Genetex GTX70255, 1:1,000) were used. The following Odyssey secondary antibodies were used: anti‐mouse, and ‐rabbit antibodies (Li‐COR, LI 926‐68070, LI 925‐32211) at 1:5,000 dilution for the final detection. For immunocytochemistry, Syndecan‐2 (SDC2, rabbit; Santa Cruz sc‐15348; 1:50) was used followed by staining with secondary antibodies conjugated with Alexa 488 (goat anti‐rabbit; Invitrogen A‐10680; 1:500).

Western blot analysis was performed to verify the expression of widely accepted exosomal markers, including CD63 (Biorbyt, orb11317; 1:500) and TSG101 (Genetex, GTX70255; 1:500). Goat anti-rabbit IgG-HRP (Santa Cruz, sc-2004) and goat anti-mouse IgG-HRP (Santacruz, SC-2005) were applied as secondary antibodies, respectively. Primary mouse monoclonal IgG1 anti-beta Actin antibody [AC-15] (abcam, AB6276) was used as an internal control. EVs (25 µg) were directly diluted 1 to 4 in sample buffer and were applied for western blot experiments following 6-min denaturation at 95 °C. SuperSignal West Femto Kit, Thermo Fisher Scientific, USA) was applied for signal detection.

The total proteins were extracted from cells or tissues using 1 × RIPA lysis buffer supplemented with a protease inhibitor cocktail (Roche). The concentrations of extracted total protein samples were measured by a BCA Protein Assay Kit (Beyotime). Equal amounts of proteins were separated by SDS-PAGE and transferred to nitrocellulose filter membranes. After blocking the membranes with non-fat milk (5% w/v) for 1 h, the proteins were incubated at 4 °C overnight with the primary antibodies against CD63 (1:1,000, WL02549, Wanleibio, China), CD81 (1:1,500, GTX101768, Gene Tex, USA), TSG101 (1:1,500, GTX70255, Gene Tex), ACSL4 (1:10,000, ab155282, Abcam, UK), GPX4 (1:1,000, A13309, Abclonal, USA), GAPDH (1:5,000, AC002, Abclonal), or β-Tubulin (1:5,000, AC021, Abclonal). Next, the membranes were incubated with secondary anti-mouse or anti-rabbit antibodies (RS23910 and RS23920, ImmunoWay, USA) in dark at room temperature for 1 h. The membranes were scanned, and the gray values of protein band were detected by Odessey CLx (LI-COR, USA).