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Fig. 1 | Cell Communication and Signaling

Fig. 1

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

Fig. 1

FAF1 is secreted via nonclassical exocytosis. a SH-SY5Y cells were transfected with vector control (VC) or 3xFlag-FAF1 plasmid. At 24 h after transfection, the culture medium was replaced with serum-free medium for the indicated times. S.E.: short exposure, L.E.: long exposure. b Rat primary neuronal cells were transduced with AAV1-hFAF1 viral vectors. At 3 days after transduction, the culture medium was replaced with serum-free neurobasal medium for 48 h. c Cells were transfected with lacZ or 3xFlag-FAF1 plasmid. At 24 h after transfection, the culture medium was replaced with serum-free medium, and cells were cultured for 24 h. d Cells were transfected with VC or 3xFlag-FAF1 plasmid. At 24 h after transfection, the culture medium was replaced with serum-free medium containing CHX (20 μg/ml) for the indicated times. e Left panel: Cells were transfected with VC or 3xFlag-FAF1 plus α-syn plasmid. At 24 h after transfection, the culture medium was replaced with serum-free medium containing DMSO (vehicle), MPP+ (1 mM), H2O2 (100 μM), or Baf A1 (50 nM) for 24 h. Right panel: The graph shows the densitometric analysis of immunoblotting of FAF1 in conditioned medium (CM) shown in the left panel (n = 3). f Upper panel: Cells were transfected with 3xFlag-FAF1 plasmid. At 24 h after transfection, the culture medium was replaced with serum-free medium, and the cells were cultured at 18 °C or 37 °C for 24 h. Lower panel: The graph shows the result of densitometric analysis of FAF1 immunoblotting in CM shown in the upper panel (n = 3). g Upper panel: Cells were transfected with 3xFlag-FAF1 plasmid. At 24 h after transfection, the culture medium was replaced with serum-free medium containing BFA (2 μg/ml) for 24 h. Lower panel: The graph shows the result of densitometric analysis of FAF1 immunoblotting in CM shown in the upper panel (n = 3). Cell lysate (CL) and concentrated CM were analyzed by western blotting with the indicated antibodies. All lanes were loaded with the same amount of total protein. The data are expressed as the mean ± S.D. of three independent experiments. Statistical comparisons were performed using using ANOVA followed by Tukey’s HSD post hoc analysis (e) and Student’s t-test (f and g). *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. = not significant

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