The AAA-ATPase VPS4 Regulates Extracellular Secretion and Lysosomal Targeting of α-Synuclein (original) (raw)
Figure 6
Part of the cellular α-synuclein was trafficked via a recycling endosome pathway for extracellular secretion.
HEK293T cells expressing Myc-αSYN were co-transfected with mock (EGFP), EGFP-wt-Rab11a, EGFP-CA-Rab11a, or EGFP-DN-Rab11a expression plasmids. At 48 hours following transfection, the cells were harvested and fractionated into cytosol, endosome, and lysosome. Fractionated samples as well as total proteins from the culture media (50 µg per lane) were subjected to immunoblot analysis using anti-Myc, anti-EGFP Abs. A successful fraction was verified by the presence of a specific marker proteins. As shown in the blot, secretion of αSYN oligomer in culture medium was partly reduced by the over-expression of GDP-locked DN-Rab11a (asterisk), accompanied by the extensive retention of HMW αSYN species in the endosome (double asterisk). Representative blots from three separate experiments are shown.