Figure 4: Transgenic hA53T versus hWT αS expressed in αS-/- mouse brain. | Nature Communications

Figure 4: Transgenic hA53T versus hWT αS expressed in αS-/- mouse brain.

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

Figure 4

(a) Whole brains from both genotypes immediately before mincing. (b) Minced brain bits from both mouse genotypes were subjected to crosslinking, and PBS-soluble (‘cytosolic’) fractions blotted (Syn1). Untreated (-), DSP/βME-treated (DSPr) and DSG-treated samples (in technical triplicate) are shown at identical short (S) exposures of hWT and hA53T samples (left panel) or a longer (L) exposure of the hA53T samples (right panel: exposure matched to αS14 intensity of hWT αS in the left panel). (c) Densitometry of the cytosolic αS60:14 ratios (relative to hWT, set to 1) based on both S and L exposures (N=3 mice of each genotype analysed on different days in triplicates of separate brain-bit samples, total n=9); NS, not significant. (d) Densitometry of cytosolic αS60 and αS14 bands in both genotypes based on identical exposures (N=3, n=9); values relative to hWT αS60. (e) DSG crosslinked mouse brain samples: cytosols blotted for αS (Syn1, 15G7, C20) and DJ-1; Ponceau-staining of the blot membrane is on left. DJ-1 served as control for equal crosslinking efficiency and equal loading. (f) Minced brain bits from both genotypes: TX-100 total homogenates (cytosolic and membrane proteins). Untreated (-), DSP/βME-treated (DSPr) and DSG-treated samples in triplicates (Syn1 mAb, right panel). Left panel, Ponceau-staining of the membrane. (g) DSG crosslinking of PBS-insoluble TX-100-soluble (TX) fractions of brain from both genotypes in triplicates. (h) Densitometry of αS60 and αS14 in the TX fractions (N=2 mice of each genotype analysed on different days in triplicates of separate brain bits, total n=6); values relative to those of hWT αS14. *P<0.05, **P<0.01; Student’s t-test (see Methods) for all quantifications shown; error bars, s.d.

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