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. 2012 Mar 7;32(10):3301–3305. doi: 10.1523/JNEUROSCI.5368-11.2012

Figure 1.

Figure 1.

αS monomers and oligomers accumulate in the ER. A, ER/Ms (P100) obtained from PreS and end-stage (sick) A53T αS Tg mice were solubilized with 0.5% TX-100 and centrifuged to obtain the supernatant (TX-S) and the insoluble pellet (TX-I). Arrowheads, SDS-stable high-MW αS. Bottom, Shorter exposure of the αS blot. B, TX-Stot and TX-Ptot fractions from the spinal cords of nTg and end-stage A53T αS Tg mice immunoblotted for αS. C, Detergent-insoluble fractions (TX-I from ER/M and TX-Ptot) from two mice immunoblotted for αS. D, ER accumulation of toxic αS oligomers. Dot-blot analysis of SpC microsomes from nTg and A53T αS Tg mice (6 months, PreS, Sick). Also analyzed are total TX-Ptot and C (detergent-free) from sick A53T αS Tg mice. Quantitative analysis of dot-blots showing values normalized for the total αS. Values are given as the mean ± SEM (n = 3–4). *p < 0.05, **p < 0.001, one-way ANOVA. E, Left, Dot-blot analysis of microsomes from diseased A53T mice (P100); in vitro assembled αS (αSF) and Tau (Tau-F) fibrils; or total insoluble fraction from Tg mice (TX-Ptot) shows FILA-1 specificity for αS oligomers. Right, Dot-blots of various microsome fractions obtained from different αS Tg mouse lines [age-matched diseased A53T αS(G2-3), A30P αS(O2), or WT αS(I2-2)]. F, Dot-blots of ER/M from different areas [α-synucleinopathy-affected areas (SpC) or disease-free areas (Ctx). Right, Dot-blot analysis of ER/M from PreS or diseased (Early, End) mice for FILA-1 or αS. G, ER-associated αS monomer but not the high-MW αS is protected from proteolysis by PK. In the presence of 0.5% TX-100, both αS and the ER-resident grp78 are completely proteolyzed by PK. H, ER/M fractions were first exposed to PK, followed by 0.5% TX-100 extraction, and were fractioned into TX-S and TX-I fractions. The αS monomers remain soluble, but proteolyzed αS fragments remain insoluble. I, Dot-blot analysis of ER/M fractions shown in H for FILA-1 or αS. In the PreS mice, the αS monomer and the oligomers are resistant to PK, indicating that they are in the ER lumen. Even in the absence of PK, TX-100 abolished both A11 and FILA-1 reactivity in PreS samples. I also shows dot-blot analysis of TX-100-soluble and -insoluble ER/M fractions without the prior PK treatment. Note that the FILA-1 signal is abolished with TX-100 in PreS mice but not in Sick mice.