Figure 3: Intact-cell crosslinking of fPD-linked αS missense mutations. | Nature Communications

Figure 3: Intact-cell crosslinking of fPD-linked αS missense mutations.

From: Parkinson-causing α-synuclein missense mutations shift native tetramers to monomers as a mechanism for disease initiation

Figure 3

(a) DSG crosslinking analysis of M17D cells transiently transfected with αS WT or the indicated mutations. Western blots for endogenous DJ-1 and transfected αS in duplicate (Syn1); each lane is one transfection. (b) Analagous to Fig. 2a, but using the reducible crosslinker DSP: upper panel, non-reduced: bottom panel, βME-reduced (Syn1). (c) DSG and DSP crosslinking, plus meta-analysis of both: intensity of αS60 alone (upper panel) or αS60+80+100 (lower panel) is graphed relative to WT αS (set to 1) (DSG: N=8 experiments each done in biological duplicates (n=2) on different days, total n=16; DSP: N=4, n=9). For better visibility in this and the following graphs, only one-direction error bars are shown. (d) DSG and DSP crosslinking plus meta-analysis: levels of αS60 alone (upper panel) or αS60+80+100 together (lower) relative to WT αS; (e) level of αS14 alone relative to WT αS. (f) Representative western blots of DSG crosslinking of 3x (H50Q-G51D-A53T) and 4x (3x+E46K) compound fPD mutants relative to G51D alone. (g) Quantification of αS60:14 ratio for 3x and 4x compound fPD mutants relative to G51D alone (N=4, n=8). *P<0.05, **P<0.01; one-sided ANOVA (see Methods) for all quantifications shown; error bars, s.d.

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