Horseradish peroxidase labeled anti mouse or rabbit igg

WB analysis was performed as described previously [85 (link)]. Equal amounts of EV fractions (15 µL (~10 µg); F1–F8) or whole-cell lysate (10 µg; prepared using RIPA buffer: ThermoFisher Scientific, Waltham, MA, USA) were solubilized using 4× Laemmli buffer (Biorad, Feldkirchen, Germany) in reducing conditions at 95 °C for 10 min. Samples were then separated by SDS-PAGE under reducing conditions, and immunoblot analyses were performed using antibodies (Supplementary Table S8) followed by horseradish peroxidase-labeled anti-mouse or rabbit IgG (Jackson ImmunoResearch). Protein bands were visualized using the enhanced chemiluminescence Western blot detection reagent (GE Healthcare) and the fusion fx Imager/fusion software (Fusion FX7 Edge 18.05, Vilber Lourmat; https://www.vilber.com/fusion-fx/).

Western blotting was carried out as previously described [26 (link)]. Briefly, RIPA buffer (R0278, Sigma-Aldrich) with a protease inhibitor cocktail (complete Tablets Mini, Roche, Basel, Switzerland) was used to isolate total protein from cells. A colorimetric test (PierceTM BCA Protein Assay Kit, Thermo Fisher Scientific) was used to determine the total protein concentration. SDS-PAGE was used to separate 10 µg of total protein under reducing conditions, and immunoblot analyses were carried out with antibodies (Supplementary Table S1) against PAX6 (clone#poly901301—1:1000; clone#D3A9V—1:1000) and GAPDH (1:50,000), followed by horseradish peroxidase-labeled anti-mouse or rabbit IgG (Jackson ImmunoResearch Europe, Ely, UK). Enhanced chemiluminescence Western blot detection reagent (GE Healthcare, München, Germany) and FUSION FX imager/fusion software (Vilber Lourmat, Collégien, France) were used to visualize protein bands.