Fig 2: Immunoblot analysis of SNARE proteins in control (Ctrl) and ALS patient ventral horns. (A) Ventral horn samples were examined for STX1A, STX1B and SNAP25 expression by immunoblotting. GAPDH was used as internal control for protein loading. (B) Histograms summarizing the densitometry analysis of the protein bands shown in A, calculated as each SNARE/GAPDH expression ratio, as indicated (n = 6 for Ctrl and n = 6 for ALS; *P < 0.002). Samples are indicated by their Bank ID as reported in Table 1. The image in A was cropped for clarity from the original in Supplementary Figure 3.

Fig 3: Specificity test for the selected nanobodies in immunofluorescence and Western Blots. (a & b) COS-7 cells transiently transfected with SNAP-25-EGFP or Syntaxin 1A-EGFP were stained with S25-Nb10 or Stx1A-Nb6 conjugated with a single Atto647N fluorophore. The nanobody signal correlates with the EGFP signal and shows no staining in untransfected cells (revealed by Hoechst-nuclear staining; white arrowheads). The scale bar represents 10 µm. Note that Syntaxin 1A accumulates in the ER-Golgi region due to an impaired export caused by the lack of neuronal cofactors, as it was found in the past30 (c & d) The following samples were loaded in a denaturating SDS-PAGE and were blotted on a nitrocellulose membrane (2 µg of each purified protein, and 20 µg of total protein for the cell or brain lysates): E. coli-purified full length SNAP-25 including a Twin-Strep-Tag (tst; 27.8 kDa), or Syntaxin 1A fused to a SUMO domain and a Twin-Strep-Tag (44.7 kDa); lysates from HEK293 cells transiently transfected with full length Syntaxin 1A-tst (HEK-Stx1A, 37.5 kDa) or SNAP-25-tst (HEK-SNAP-25, 27.8 kDa); whole rat brain and rat primary hippocampal neurons (15 days in vitro). Detection was performed using S25-Nb10 (c) or Stx1A-Nb6 (d) conjugated to a single Atto647N. Both candidates specifically detect the bands at the expected molecular weights (displayed in the protein schematics), while showing no cross-reactivity to the opposite antigen or to any other proteins present in the lysates.

Fig 4: Rough mapping of the binding epitopes of the selected nanobodies. Full length antigen or truncated versions were produced in E. coli and were spotted on a nitrocellulose membrane in equimolar amounts. Bovine serum albumin (BSA) was used as negative control. (a) S25-Nb10 and (b) Stx1A-Nb6 were directly labeled with a single Atto647N fluorophore on their C-terminus and were used for protein detection. The schematics display the location and size of the truncated epitopes (indicated by numbers below). For orientation, molecular structures of the full length and the different truncated domains are displayed nearby.

Fig 5: Both nanobodies reveled epitopes that are not reported by antibodies in highly crowded regions of endogenously-expressing PC12 cells or over-expressing COS-7 cells. (a) PC12 cells were co-stained with conventional monoclonal antibodies and with our fluorescently labeled nanobodies. Monoclonal anti SNAP-25 and Syntaxin 1A (clone 71.1 and HPC-1) were detected with a secondary antibody conjugated to Abberior-Star580 (in green). S25-Nb10 or Stx1A-Nb6 were conjugated to a single Atto647N fluorophore (in red). Laser scanning confocal images of the nanobody and the antibody signals colocalize relatively good at the plasma membrane, but the nanobodies also reveal stronger signals in the perinuclear areas (especially evident for SNAP-25). The scale bar represents 10 µm. (b) Laser scanning confocal images of COS-7 cells transiently transfected with SNAP-25 or Syntaxin 1A fused to EGFP (in green) and co-stained with monoclonal primary and Cy3-fluorescently labeled secondary antibody (clone 71.1 and clone 78.2, in magenta) and nanobodies directly conjugated to Atto647N (in red). Scale bar represents 10 µm. (c) Line profiles of the white lines displayed in (B) are shown for all three channels, each channel was normalized to its maximum signal intensity on the picture. (d) The average fluorescence of full cells, black bars or at the perinuclear regions, light-grey bars in respect to their EGFP signals were calculated. For every selected region of interest, the averaged fluorescence signal of antibodies or nanobodies was calculated and normalized to the average EGFP-fluorescence in the respective region. Statistical analysis was done using unpaired t-test, error bars represent SEM, from three independent experiments analyzing a total of 37 cells for SNAP-25-EGFP and 36 cells for Stx1A-EGFP. n.s. = not significant.