Fig. 2

Knockdown of SCAMP5 decreases dopamine level and synuclein secretion via exosome pathway. A PC12 cells were transfected with plasmids and siRNA containing as indicated. At 72 h after transfection, cell lysates (CL) were harvested and analyzed by Western blot. CON denotes the control group. The Myc-SCAMP5 expression vector counteracts si-Scamp5 interference. The primary antibody was anti-SCAMP5. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a loading control. B PC12 cells were co-transfected with plasmids and siRNA as indicated. At 72 h after transfection, dopamine concentration in cell culture supernatant was analyzed by ELISA. Human wild-type SCAMP5-WT (SCAMP5-WT), but not SCAMP5-R91W, could restore the dopamine in cell culture supernatant after Scamp5 knockdown in PC12 cells. C PC12 cells were co-transfected with plasmids and siRNA as indicated. At 72 h after transfection, extracellular α-synuclein concentration was analyzed by ELISA. Human SCAMP5-WT, but not SCAMP5-R91W, could restore the extracellular α-synuclein in cell culture supernatant after Scamp5 knockdown in PC12 cells. D, E SH-SY5Y cells stably expressing GFP-α-synuclein were transfected with siRNA as indicated. At 72 h after transfection, culture medium and CL were harvested and analyzed by Western blot. D Lysates were analyzed with the indicated antibodies. The primary antibody was anti-SCAMP5, anti-GFP. GAPDH served as a loading control. E Quantitative comparison of GFP-α-synuclein in cell medium of SH-SY5Y cells. CM cell medium, CL cell lysates. The GFP-α-synuclein in cell medium was reduced after SCAMP5 knockdown in SH-SY5Y cells. F, G SH-SY5Y cells stably expressing GFP-α-synuclein were transfected with plasmids and siRNA as indicated for 72 h, followed by the collection of cell medium and cell lysates for Western blot analysis. F Lysates were immunoblotted with indicated antibodies. The primary antibody was anti-SCAMP5, anti-GFP. GAPDH served as a loading control. G Quantitative comparison of GFP-α-synuclein in cell medium of SH-SY5Y cells. Human SCAMP5-WT, but not SCAMP5-R91W, could restore the reduced GFP-α-synuclein in cell medium after SCAMP5 knockdown in SH-SY5Y cells. H, I SH-SY5Y cells stably expressing GFP-α-synuclein were transfected with siRNA as indicated, and harvested at 72 h post-transfection. Protein levels were assessed by Western blot. The loading/total volumes of CL, CM, EXO, and FT were 2μL, 40μL, 10μL, 20μL respectively. H Lysates were probed with indicated antibodies. The primary antibody was anti-GFP, anti-Calnexin and anti-CD63. GAPDH served as a loading control. I Quantitative comparison of GFP-α-synuclein in exosomes of SH-SY5Y cells. The GFP-α-synuclein in exosome was reduced after SCAMP5 knockdown in SH-SY5Y cells. CL: cell lysates, CM: cell medium, EXO: exosome, FT: flow through. CD63: exosome marker; Calnexin: ER marker. In panels B, C, G and I data are presented as mean ± SEM. n ≥ 3 independent experiments. Significance levels are denoted as follows: *P < 0.05, **P < 0.01, ***P < 0.001