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. 2011 Jan;25(1):326–336. doi: 10.1096/fj.10-164624

Figure 1.

Figure 1.

αSyn oligomers are secreted from cells. A) Principle of bioluminescent-protein complementation assay based on G. princeps luciferase, adapted from Kerppola (56) B) Intact cells and CM from H4 cells cotransfected with S1/S2 were assayed for luciferase activity 48 h post-transfection. Intact cells displayed a 6-fold increase in luciferase activity compared to control. CM had a 26-fold increase in luciferase activity vs. control. n = 4. C) β-Gal activity is not significantly (ns) increased in medium compared to cells when cotransfected with S1/S2. n = 6, P = 0.78. D) Ex vivo mixing of S1 CM with S2 CM leads to a time-dependent increase in luciferase activity. Data are representative of 3 independent experiments. E) Ex vivo aggregation is not facilitated by luciferase tags. No luciferase activity was detected when S1 CM was mixed with Aβ-luc2 CM or S2 CM was mixed with Aβ-luc1 CM for 3 d. By contrast, a significant increase in luciferase activity was detected when S1 CM was incubated with S2 CM for 3 d. n = 6. F) αSyn protein concentration in CM of S1/S2- wt αsyn- or mock-transfected cells. n = 3. G) Dot blot of CM from wt αsyn-transfected H4 cells using anti-synuclein Syn-1 or oligomer-specific antibody A11. Nontransfected medium was used as negative control. Representative blots from 4 independent experiments. Data represent means ± sd. **P < 0.01; ***P < 0.001.