Fig. 1

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)