Fig. 2
From: Recycling of endocytic BDNF through extracellular vesicles in astrocytes
EV-mediated release of endocytosed BDNF from astrocytes. (A) Schematic diagram of the experiment for detecting BDNF-EGFP derived from Neuro2A cells in astrocytic EVs. (B) Dot blot showing EGFP expression in cell lysates from nontransfected (non. ; negative control) or BDNF-EGFP-transfected Neuro2a cells. (C) Dot blotting was used to detect protein expression in cell lysates and EVs extracted from the media of astrocytes treated with ATP or vehicle after being supplied with media from nontransfected or BDNF-EGFP-transfected Neuro2a cells. Antibodies targeting general markers of the endoplasmic reticulum (ER; an EV-negative marker, calregulin) or EVs (TSG101) were used. (D) Representative QD-BDNF kymographs from astrocytes were generated via ImageJ/FIJI and obtained after treatment with vehicle or GW4869 (20 µM) for 1 h prior to time-lapse imaging. Red bar: ATP (100 µM) treatment. Arrow heads: disappearance of QD-BDNF fluorescence. (E) Average percentages of ATP-induced QD-BDNF secretion events from vesicles in astrocytes pretreated with either vehicle or GW4869. ****P < 0.0001. N = 9–11 cells (vehicle = 201; GW4869 = 468 QD particles). (F) Schematic diagram showing overall process of immunocapture of CD63-positive EVs released from ACM using anti CD63-linked magnetic beads. (G-H) Bar graphs depict average concentration of BDNF from ACM using ELISA assay with anti-BDNF (G) or BDNF-EGFP with anti-GFP (H). N = 4 ACM batches derived from different astrocyte cultures. Each dot indicates the result measured from individual batch.
