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. 2012 Aug 30;2(2):358–371. doi: 10.1016/j.celrep.2012.06.012

Figure 3.

Figure 3

SERF1a Does Not Affect Nonamyloid Protein Aggregation

(A) An excess of SERF1a (open green square) was unable to convert stable dopamine-induced “off-pathway” aSyn aggregates (closed blue square) (723 μg/ml, which is the mass equivalent of 50 μM monomeric aSyn) into amyloid fibers. As a control, the amyloid growth of 50 μM monomeric aSyn in the absence (closed blue circle) and in the presence (open green circle) of 50 μM SERF1a was measured.

(B) TEM images show no morphological difference for “off-pathways” aggregates before (left) and after agitation in the presence of SERF1a (right). Scale bars = 100 nm.

(C) SERF1a was not able to disassemble “off-pathway” aggregates into monomers: equimolar amounts of aggregates and SERF1a were incubated at 37°C; 10 μl aliquots were drawn and analyzed by SDS-PAGE and immunoblotting (anti-aSyn). SDS-resistant, high-molecular size aSyn oligomers were visible and stable over the whole experimental time range (20 days) despite the presence of SERF1a, which did not cause their disruption into monomeric aSyn. Lane c = control mixture of monomeric and oligomeric aSyn.

(D) SERF1a did not affect actin polymerization: the organized assembly of 10 μM actin, covalently attached to the fluorophore pyrene, was monitored by a polymerization-induced fluorescence change in the absence (closed red square) and in the presence of increasing concentrations of SERF1a (open blue circle, open upward green triangle, and open downward brown triangle).

(E and F) SERF1a did not influence unspecific protein aggregation: an excess of SERF1a (open green square) did not affect (E) heat-induced aggregation of citrate synthase (43°C) (closed blue circle) or (F) reductive aggregation of insulin (closed blue circle), as measured by changes of the solution's turbidity.

The lower graphs display changes in ThT fluorescence intensities for (D), (E), and (F), showing that ThT-reactive species did not accumulate during these reactions.

See also Figure S2. The error bars are mean averages, including SEM of three independent experiments.