Supplementary Figure 1: Biochemical characterization of TDP-43 segments.
From: Cryo-EM structures of four polymorphic TDP-43 amyloid cores
a, SDS-PAGE shows the cleavage of SegA and SegB A315E. SUMO-tagged segments are purified with Ni-column, and mixed with protease UPL1 to remove SUMO tag (0 h). After 1 h of cleavage (1 h), bands corresponding to SUMO tag alone and TDP-43 segments (indicated by arrows) are shown on the gel, indicating successful cleavage. b, c, Negative stain electron microscope (EM) images of fibrils formed by TDP-43 segments (b) without heating or (c) heated before EM sample preparation. Fibrils of mCherry-FUS-LCD with or without heating were also observed with EM as a control. (Scale bar = 200 nm) Notice that the amount of fibrils in each image does not necessary corresponding to the total amount of fibrils in each sample, since the distribution of fibrils on EM grids was not even, especially for clumped fibrils. d, Aggregation assays of the pathological fragment TDP-CTF (208–414). Wild type (WT) and mutant TDP-CTFs conjugated with SUMO tags were incubated at 4 °C overnight. Samples were separated into supernatant (s) and pellet (p) by centrifugation and analyzed by SDS-PAGE. Notice that TDP-CTF A326W and R293E-A315R behave similarly as TDP-CTF WT, whereas the other two mutants show reduced aggregation. e–g, ThT aggregation curves of TDP-43 segments with or without seeding. Data are shown as mean ± s.d., n = 4 independent experiments.
