Figure 1. Anti-C9RANT immunoreactivity is specific to c9FTD/ALS.

(A) Schematic representation of the possible protein products generated by RAN translation of expanded GGGGCC repeats in the three alternate reading frames. (B, C) Immunoreactivity of each anti-C9RANT antibody (Rb5822 and RB5823) towards indicated peptides was measured by adsorbing peptides onto carbon electrodes in 96-well MSD plates, and co-incubating wells with anti-C9RANT and a SULFO-tagged anti-rabbit secondary antibody. Anti-C9RANT binding to respective peptides was quantified by measuring the intensity of emitted light upon electrochemical stimulation of the plate using the MSD Sector Imager 2400. The amino acid sequence for (GX)₅ is Gly-Met-Gly-Asp-Gly-Ser-Gly-Leu-Gly-Thr. (D) Western blot analysis of cerebellar tissue urea fractions from C9ORF72 repeat expansion and non-expansion FTLD cases using anti-C9RANT. Note the high molecular weight product (Arrow). (E) Anti-C9RANT immunoreactivity in cerebellar urea fractions from FTLD-TDP and ALS cases with or without expanded GGGGCC repeats, as assessed by dot blot. Each dot represents one case. See also Figure S1. (F-I) Immunohistochemistry with each anti-C9RANT antibody revealed that abundant neuronal inclusion in the cerebellum of c9FTD (F, H), but not in sporadic FTLD-TDP (G, I). C9RANT-immunoreactive lesions were granular neuronal cytoplasmic inclusions (seen clearly in the Purkinje cell shown in inset of F, H). Scale Bar=50 μm in main images, 20 μm in insets.