FIGURE 4.

Involvement of CMA in degradation of ASYN in SH-SY5Y cell lines. A, generation of stable inducible Tet-Off SH-SY5Y cell lines overexpressing ΔDQ/WT and WT ASYN. Cells were treated with or without 3 μg/ml dox for 4 days, and lysates were used for ASYN immunoblotting. B and C, overexpressed ΔDQ ASYN displays a slower rate of degradation compared with WT ASYN. B, ΔDQ/WT and WT ASYN SH-SY5Y cell lines were cultured in the absence of dox for 5 days, and then dox was added at the indicated times. Left, representative immunoblot of ASYN levels. Right, quantification of rate of turnover of overexpressed ΔDQ/WT and WT ASYN after dox addition. C, ΔDQ/WT and WT ASYN SH-SY5Y cell lines were cultured in the absence of dox for 5 days. The cells were then labeled with [³⁵S]cysteine/methionine for pulse-chase. ASYN was immunoprecipitated with C20 ASYN Ab, and its levels were assessed by autoradiography. ERK immunoprecipitation was used as negative control (Ctrl). Left, representative pulse-chase is shown. Right, quantification of the turnover of ΔDQ/WT and WT ASYN. All results are expressed as the ratio of OD values to the corresponding controls, and all data are presented as a percent of each time point compared with the value at time point 0. (*, p < 0.05; **, p < 0.01; ***, p < 0.001, Student's t test comparing ΔDQ/WT to WT ASYN).