Figure 2.

The E46K mutant α-syn is degraded by a proteasome and a macro-autophagy pathway. (A) PC12 cells stably overexpressing the E46K mutant α-syn were treated with 10 μM MG132 for 24 h. Cells were lysed and subjected to SDS-PAGE using an anti-α-syn antibody. (B) Stable PC12 cells overexpressing E46K mutant α-syn were treated with 10 μM Lac for 24 h. α-Syn levels were assessed by using an anti-α-syn antibody. (C) Cells were treated with 10 μM MG132 or 10 μM Lac for 24 h and mRNA levels of the E46K mutant α-syn were detected by using RT-PCR. (D) Cells were labeled with Lyso-Tracker for 20 min and visualized by using a confocal microscopy. Representative images were shown. Scale bar: 10 mm. (E) Cells were treated with 100 μM CQ, 30 μg/mL HCQ, and 20 mM NH₄Cl for 24 h. Cells were lysed and subjected to SDS-PAGE using an anti-α-syn antibody. (F) Cells were incubated with 10 mM 3-MA, 20 mM NH₄Cl, and a combination of 3-MA and NH₄Cl for 24 h. Cells were lysed and subjected to immunoblot analysis using an anti-α-syn antibody. (G) Cells were treated with 100 μM CQ, 30 μg/mL HCQ, 20 mM NH₄Cl, 10 mM 3-MA, and a combination of NH₄Cl with 3-MA for 24 h. RT-PCR was conducted to assess the mRNA levels of E46K mutant α-syn. (H) PC12 cells were treated with 100 mM Tre and 200 nM Rap for 24 h and α-syn levels were assessed using Western blotting. (I) PC12 cells were treated with Rap for indicated concentration and harvested for immunoblotting for α-syn. (J) PC12 cells were treated with 400 nM Rap for the indicated periods of time and cell extracts were subjected to the immunoblot. Representative blots of three independent experiments were shown. β-actin levels demonstrate equal loading. (K) Cells were treated with 100 mM Tre of 400 nM Rap for 24 h and mRNA levels of E46K mutant α-syn were detected by RT-PCR. Data were expressed as means ± SD for three independent experiments performed in triplicate. * p < 0.05, ** p < 0.01 compared with the control group.