Figure 2: Intact-cell crosslinking of αS expressed at varying levels. | Nature Communications

Figure 2: Intact-cell crosslinking of αS expressed at varying levels.

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

Figure 2

(a) DSG-crosslinking analysis of an αS DNA gradient (0-8 μg per 6-cm culture dish). Western blots for αS (Syn1) and endogenous DJ-1. (b) DSG samples: αS60 intensities plotted against αS14 (densitometry). Highest value in each series was set to 1; graph shows mean data for N=5 independent experiments of 1, 2, 3, 4, 6, 8 μg DNA (2 exps.), 2, 4, 8 μg DNA (1 exp.) and 4, 8 μg DNA (2 exps.); data points generated in the same experiment are indicated by identical symbols. (c) αS60:αS14 ratios for the same samples as in 2b. (d) DSP-crosslinking analysis of an αS DNA gradient (0; 1-8 μg); western blots (Syn1) for αS in non-reduced and βME-reduced samples (e) Quantification of DSP samples: αS60 versus αS14. Highest value in each series was set to 1; N=3 independent experiments of 1, 2, 3, 4, 6, 8 μg DNA (1 exp.), 2, 4, 8 μg (1 exp.) and 4, 8 μg (1 exp.). (f) αS60:14 ratios for the same samples as in (e). (g) ELISA analysis of αS DNA gradients (1, 2, 4, 8 μg) transfected into M17D cells compared to human cortical homogenate (PBS fraction). Below: western blots for 0 and 8 μg DNA transfection versus human brain homogenate (red). N=2 for gradients (different days, different DNA preps) and brain homogenates. (h) ELISA of αS WT and fPD mutant transfectants (8 μg per 6-cm dish), PBS fraction. Graph: concentrations versus αS WT set to 1 (N=10 independent transfections on 4 different days using at least 4 different DNA preps per αS variant). tf’ed, transfected. (i) ELISA of αS WT and fPD mutant transfectants after 1 mM DSG crosslinking and sequential extraction (PBS→PBS/1%Triton→2% LDS→88% formic acid=FA). Graph: concentrations relative to the PBS fractions of the respective αS variant set to 1.

Back to article page