Image quant las 100 mini

ARPE‐19 cells, treated or control, were collected in RIPA Buffer (Thermo Fisher Scientific) and a protease/phosphatase inhibitor cocktail (Sigma‐Aldrich). Equal amounts of protein (30 µg) were loaded and measured by SDS‐PAGE on 4%‐12% SDS‐Polyacrylamide gel electrophoresis and electro‐blotted onto a polyvinylidene difluoride membrane (PVDF; Millipore) by wet transfer. Membranes were incubated overnight with antibodies against VEGFA (1:200; Santa Cruz Biotechnology) and ß‐actin (1:1,000; Santa Cruz Biotechnology) as loading control. Finally, the membranes were incubated for 2 hours at room temperature with anti‐mouse and anti‐rabbit IgG‐HRP antibodies (Santa Cruz Biotechnology). The visualization was performed with ECL (Pierce, Thermo Fisher Scientific) and detected with Image Quant LAS‐100 mini (GE Healthcare). Protein levels were quantified by densitometry using ImageJ software (NIH).

sEVs, treated or control, were resuspended in RIPA-50 buffer (Thermo Fisher Scientific, Waltham, MA, USA) and protease/phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Equal amounts of protein (70 µg) were loaded and measured by SDS-PAGE on 4–12% SDS-Polyacrylamide gel electrophoresis and electro-blotted onto a polyvinylidene difluoride membrane (PVDF; Millipore, Burlington, MA, USA) through wet transfer. After blocking with 5% skim milk, membranes were incubated overnight with CD9 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and CD63 antibodies (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Finally, membranes were incubated for 1 h at room temperature with anti-mouse and anti-rabbit IgG-HRP antibodies (1:500 Santa Cruz Biotechnology, Santa Cruz, CA, USA). The visualization was performed with ECL (Pierce, Thermo Fisher Scientific, Waltham, MA, USA) and detected by Image Quant LAS-100 mini (GE Healthcare, Chicago, IL, USA).