Extended Data Fig. 4: Disrupting the interface between α-Syn and VAMP2 impairs the catalysis role of α-Syn in SNARE complex assembly.
From: VAMP2 chaperones α-synuclein in synaptic vesicle co-condensates
Expression levels of three SNARE proteins in HEK293T cells co-transfected with plasmids expressing (a, top; c, top; and d, middle) syntaxin-1, (a, middle; c, middle; and d, right) SNAP-25, and (a, bottom; c, bottom; and d, left) VAMP2 at a 1:1:1 ratio, together (a,b) with an increasing amount (0-3-fold) of α-Syn variant plasmids (WT and α-Syn100), and (c,d) with 4-fold expression plasmids of GFP, α-SynWT and α-Syn5K. Cell lysates were immunoblotted for the indicated SNAREs, followed by quantitation for representative immunoblots (see Fig. 4e). Total expression levels of individual SNARE proteins or balancing pCMV5-emerald in transfected HEK293T cells were analyzed by melting SNARE-complexes in SDS sample buffer (100 °C for 20 min), and quantitated by immunoblotting, normalized to α-tubulin (α-Tub) or heat shock cognate 71 kDa protein. HEK293T cell lysates (e) (n = 6 independent replicates) or primary neuron lysates (f) (n = 5 independent replicates) were immunoblotted for SNARE complexes and α-Syn followed by quantification and normalization to heat shock cognate 71 kDa protein (Hsc70) or β-tubulin-III (Tuj1) levels. Data shown are means ± SD; unpaired two-tailed t-test; n = 3 (a,b) or 6 (d) independent cultures. Source numerical data and uncropped blots are available in the Source Data Extended Data Fig. 4.
