The AAA-ATPase VPS4 Regulates Extracellular Secretion and Lysosomal Targeting of α-Synuclein (original) (raw)
Figure 7
Increased secretion of α-synuclein by DN-VPS4A is restored by DN-Rab11a.
GDP-locked DN-Rab11a strikingly restored the hypersecretion of αSYN triggered by the impaired MVB sorting pathway (closed triangle). HEK293T cells co-expressing Myc-αSYN and 3XFLAG-DN-VPS4A (A) and SH-SY5Y neuronal cells expressing 3XFLAG-DN-VPS4A (B) were further transfected with EGFP-DN-Rab11a. Forty eight hours post transfection, the cells were harvested and solubilized in RIPA buffer. Whole cell lysates as well as total proteins from culture media (50 µg per lane) were then subjected to immunoblot analysis using anti-Myc, anti-synuclein-1, anti-FLAG, and anti-EGFP Abs. Hsp90 and BSA were used as markers for the cytosol and culture medium, respectively. Asterisk indicates unspecific band. Representative blots from three independent experiments are presented.