Fig 2: Basic characterization of novel monoclonal antibodies against C9orf72. a Schematic representation of postulated human and murine C9orf72 protein isoforms with epitopes recognized by novel monoclonal antibodies (mAbs) against C9orf72. In humans, two C9orf72 protein isoforms are postulated with isoform 1 representing a 481 amino acid protein, also known as long isoform or C9-L (transcribed by transcript variant 2 with the GGGGCC repeat located in the promoter region and transcript variant 3 with the GGGGCC repeat located in the first intron); and isoform 2 representing a 222 amino acid protein, also known as short isoform or C9-S (transcribed by transcript variant 1 with the GGGGCC repeat located in the first intron). In mice, three protein isoforms are postulated, with isoform 1 corresponding in size to human C9-L with 98% similarity on amino acid sequence. The red lines in the murine isoforms illustrate two amino acid changes between the human and mouse C9orf72 sequence in the epitope recognized by mAbs 5F6 and 12G10. b Immunoblot analysis of protein lysates of HEK293 cells expressing untagged or myc-DDK-tagged human C9-L and C9-S or myc-DDK-tagged murine C9orf72 isoform 1 (mC9–1) with novel C9orf72 mAbs. Clones 12E7 and 1C1 recognize hC9-S and hC9-L as well as mC9–1. Clones 5F6 and 12G10 specifically recognize human but not mouse C9orf72. Clones 2H7 and 15C5 specifically recognize an epitope in the C-terminus only present in hC9-L and mC9–1 but not hC9-S, however, both mAbs also recognize an unspecific band (asterisk). c Double label immunofluorescence for anti-myc (green) and anti-C9orf72 (red) of HEK293 cells transiently expressing myc-DDK-tagged hC9-L, hC9-S or mC9–1 confirms the specificity of the indicated mAbs for specific C9orf72 isoforms or species. Hoechst 33342 staining of nuclei (blue) in the merged images. Scale bar: 20 µm. d Immunoblot analysis of total protein lysates from brains of wild-type (C9+/+) and C9orf72 knock-out (C9-/-) mice. Only a single band around 50 kDa corresponding in size to the murine isoform 1 is detected with mAbs 12E7 and 1C1 in wild-type mice (arrowhead). Note, that this band is completely absent in C9-/- mice, validating the high specificity for C9orf72 of the mAbs 12E7 and 1C1. The weak band labeled with an asterisk seen in C9-/- with the mouse mAb 1C1 represents mouse IgG heavy chain recognized by the anti-mouse IgG (H + L) detection antibody (see Additional file 1: Figure S1b for secondary antibody control). GAPDH is shown as loading control. MW size marker: Precision Plus Protein Dual Color Standards (b and d)

Fig 3: C9orf72 co-localizes with synaptic vesicles in human iPSC derived motor neurons. a C9orf72 positive puncta (green) are seen in the axons of 30 day old human iPSC derived motor neurons which consistently co-localize with SMCR8 (red, upper panel) and partially co-localize with LAMP2 as lysosomal marker (red, middle panel) or with the synaptic vesicle marker synaptophysin (red, lower panel). Nuclei are stained with DAPI (blue) in the merged images. C9orf72 labeled with 12E7 antibody in the upper panel and with 1C1 in the middle and lower panel. b Graph showing the percentage of C9orf72 positive puncta co-localizing with SMCR8, LAMP2 or synaptophysin. Values are shown as mean ± SD

Fig 4: Protein interaction analyses by Western blotting and co-IP of the SMCR8 complex in HEK 293T cells (see Fig. S1 for antibody analyses). a Endogenous C9-L (arrow) co-IPs with FLAG-tagged SMCR8. The thick arrowhead marks a band consistent in size with C9-S. b FLAG-tagged C9orf72 co-IPs both endogenous and co-transfected HA-tagged SMCR8. c FLAG-tagged WDR41 protein co-IPs both endogenous C9orf72 and SMCR8 proteins (indicated by arrows). Tagged C9orf72 and WDR41 proteins of (b) and (c) are not visible in whole cell lysates at the Western blot film exposure times shown. d C9orf72-FL, FL-SMCR8, and empty vector were immunoprecipitated on a-FLAG agarose from transfected 293T whole cell lysates, resolved on a polyacrylamide gel, and silver-stained. IP reactions were in the presence or absence of 50 µg/ml RNases. Complex immunoprecipitate samples were analyzed by MS sequencing. Arrows indicate full-length protein bands. Protein molecular weight markers are those of Novex Sharp Pre-stained Protein Standard (Thermo Fisher Scientific)

Fig 5: BV-2 cells harboring the C9orf72 hexanucleotide repeat expansion do not form intranuclear sense RNA foci. Cells were transiently co-transfected with 2R or 66R in combination with a plasmid coding for ZsGreen1 (green). Nuclei were stained with DAPI (blue). Sense RNA foci were detected via FISH using TYE 563-(CCCCGG)3 LNA probes (red) in N2a cells transfected with 66R plasmid (A) but not in N2a cells transfected with 2R plasmid (C). No fluorescent signal was detectable when using TYE 563-(CAG)6 negative control probes, demonstrating the specificity of TYE 563-(CCCCGG)3 probes (B). Sense RNA foci were not detected in BV-2 cells transfected with 66R plasmid 24 or 48 h after transfection (D,E). Representative images from three (D) and two (E) independent cell culture experiments are shown. Scale bar = 10 µm (A–E); scale bar = 2 µm (A, right picture). DAPI, 4',6-diamidino-2-phenylindole; FISH, fluorescence in situ hybridization; LNA, locked nucleic acid; ZsGreen1, Zoanthus sp. green fluorescent protein 1.