Figure 2

3B12A intrabodies selectively interact with misfolded and/or mislocalised TDP-43. (a,b) Confocal laser micrographs of HEK293A cells expressing TDP-43-FLAG (red) and Myc-tagged 3B12A intrabodies (green) at 48 h after transfection. DAPI was used for counterstaining of nuclei (blue). Scale bar = 20 µm. (c) 3B12A scFv (VH_VL or VL_VH) interacted with cytoplasmic TDP-43 (TDP-43^mNLS) and aggregated TDP-43 (TDP-43^C173S/C175S and TDP-43^{mNLS,C173S/C175S}). DCS indicates the C173S/S175S mutant. (d) TDP-43^C173S/C175S, TDP-43^{mNLS,C173S/C175S} and TDP-43^mNLS were also immunoprecipitated with both 3B12A scFv and 3B12A nanobodies (VH or VL). (e) Sandwich ELISAs showing the specific immunoreactivity of 3B12A intrabodies against TDP-43 species. Lysates from HEK293A cells co-transfected with Myc-tagged 3B12A intrabodies and FLAG-tagged TDP-43 or SOD1 (WT or G93A mutant) were reacted to ELISA plates previously coated with anti-FLAG antibody and subsequently detected with anti-Myc antibody. Untagged-TDP-43^{mNLS, C173S/C175S} was used as a negative control. (f) Sandwich ELISA showing the immunoreactivity of 3B12A scFv for the familial ALS-linked TDP-43 mutants A315T or Q331K.