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Fig. 2 | Cell Communication and Signaling

Fig. 2

From: A novel function of FAF1, which induces dopaminergic neuronal death through cell-to-cell transmission

Fig. 2

FAF1 is released to the extracellular space via both exosomal and nonvesicular pathways. a SH-SY5Y cells plated on 150 mm dishes were transfected with VC or 3xFlag-FAF1 plasmid. At 24 h after transfection, the culture medium was replaced with exosome-depleted medium, and the cells were cultured for 48 h. Then, exosomes were isolated from the CM by ultracentrifugation. CL and isolated exosomes (EXOs) were analyzed by western blotting with the indicated antibodies. CALR: calregulin. b Purified exosomes were characterized using nanoparticle tracking analysis. c Representative TEM images of exosomes are shown. Scale bar: 200 nm (left) or 100 nm (right). d The purified exosomes were treated with Na2CO3. After centrifugation at 50,000g, the integral membrane proteins were pelleted (memb.), and nonintegral and luminal proteins remained in the supernatant (sol.). These fractions were analyzed by western blotting with the indicated antibodies. e The purified exosomes were incubated with PK (2 μg/ml) in the absence or presence of 1% Triton X-100 (TX). +Tx, with 1% TX; −Tx, without TX. f Immunogold labeling of purified exosomes with anti-CD63 antibodies (left) and anti-FAF1 antibodies from VC-transfected (middle) or 3xFlag-FAF1-transfected (right) cells. Scale bar: 100 nm. g Cells were transfected with VC or 3xFlag-FAF1 plasmid. At 24 h after transfection, the culture medium was replaced with serum-free medium, and the cells were cultured for 24 h. After the CM was isolated by ultracentrifugation, the supernatant (SUP) and pellet (EXO) were analyzed by western blot with the indicated antibodies. h After immunoprecipitation of FAF1 from CM with anti-FAF1 monoclonal antibody, the immunodepleted CM was concentrated and analyzed by western blotting with the indicated antibodies. All lanes were loaded with the same amount of total protein

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