MANF Inhibits α-Synuclein Accumulation through Activation of Autophagic Pathways

Figure 3

MANF inhibited the accumulation of SNCA^WT in PD cellular model by CMA activation. (a)–(d) Effects of MANF on the levels of SNCA^WT, Lamp-2A, and Hsc70. SNCA^WT SH-SY5Y cells were treated with MANF (250, 500 ng/ml) and Dox (600 ng/ml) for 24 h (a, b) or 48 h (c, d), respectively; then, the protein levels were detected by western blot analysis. (e, f) Effects of MANF on the interaction between SNCA^WT and Hsc70. SNCA^WT SH-SY5Y cells were treated with MANF (500 ng/ml) and Dox (600 ng/ml) for 24 h. Cell lysates were immunoprecipitated with anti-SNCA, and the precipitated proteins were analyzed by immunoblotting with anti-Hsc70. (g, h) SNCA^WT SH-SY5Y cells were treated with Lamp-2A RNAi 1-3 for 48 h. The level of LAMP-2A was detected by western blot analysis. (i, j) Lamp-2A knocked down partly abolished MANF-induced SNCA^WT clearance. SNCA^WT SH-SY5Y cells were treated with Lamp-2A RNAi 2 for 24 h, followed by the incubation of MANF (500 ng/ml) and Dox (600 ng/ml) for another 24 h. The levels of LAMP-2A and SNCA^WT were detected by western blot analysis. Data were expressed as from three independent experiments. ^## vs. control group; ^$ vs. NC group; and vs. Dox-treated group; ^& vs. combined treatment with negative control Lamp-2A RNAi plasmid, MANF, and Dox group.

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