Figure 5: A53T versus WT αS in hESC- and hiPSC-derived neurons. | Nature Communications

Figure 5: A53T versus WT αS in hESC- and hiPSC-derived neurons.

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

Figure 5

(a) Crosslinking analysis of non-induced (-Dox) and induced (+Dox) hESC-derived NPCs expressing WT or A53T αS: PBS-soluble fraction. DSP/βME-treated (DSPr) and DSG-treated samples were blotted for DJ-1 and αS (Syn1). (b) Quantification of the cytosolic αS60:14 ratios (N=3 different cultures, each analysed in parallel in duplicates, total n=6); ratios relative to WT αS. (c) Quantification of cytosolic αS60 and αS14 for both genotypes (normalized to WT αS60); NS, not significant. (d) Crosslinking analysis of neurons differentiated from human A53T iPSCs versus their genetically corrected isogenic WT line (WTcorr). PBS and TX-100 fractions of untreated (−), DSP/βME-treated and DSG-treated cells were probed for DJ-1 and αS (2F12). (e) DSG-crosslinked samples: cytosols blotted for αS (2F12, C20, Syn1) and DJ-1; * non-specific band detected only by Syn1 (ref. 9). (f) Densitometry of the cytosolic αS60:14 ratios (relative to WT) as detected by 2F12, C20 and Syn1 (N=4 cultures grown independently and analysed on different days in biological duplicates or triplicates; total n=10). (g) Densitometry of cytosolic αS60 (relative to WT) for both genotypes. (h) Densitometry of cytosolic αS14 (versus WT) for both genotypes. (i) Crosslinking analysis of neurons differentiated from WT hESCs or from a genetically engineered isogenic E46K line (E46Keng). TX-100 total protein lysates of DSG-treated cells were probed with casein kinase 1α (cytosolic monomer), DJ-1 (cytosolic dimer), VDAC (membrane-associated dimer) and αS (mAbs 2F12 and Syn1). The five panels on the left are from one experiment (N#1), the three panels on the right show the Syn1 western blots from three independent experiments (N#2-4, each quality-controlled by DJ-1 and VDAC western blots). *non-specific band detected only by Syn1 (ref. 9). (j) Densitometry of the cytosolic αS60:14 ratios (relative to WT) as detected by Syn1 mAb (N=4 cultures grown independently). (k) Densitometry of cytosolic αS60 (versus WT) for both genotypes. (l) Densitometry of cytosolic αS14 (versus WT) for both genotypes. *P<0.05, **P<0.01; Student’s t-test (see Methods) for all quantifications shown; error bars, s.d.

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