Fig 1: Amperometric responses measured with the dual immunosensor for 0- (white bar) and 10-ng mL−1 BAFF or 4-ng mL−1 APRIL (grey bar) standards prepared in absence and in the presence of 1 mg mL−1 hIgG, 50 mg mL−1 HSA, 5 mg mL−1 BSA, 5 mg mL−1 HB, 30 pg mL−1 NfL, 5 pg mL−1 TAU, 1 ng mL−1 TDP-43, 500 ng mL−1 CDH-17, 10 ng mL−1 IL13Rα2, 100 pg mL−1 TNFα, 4 ng mL−1 APRIL, or 1 ng mL−1 BAFF
Fig 2: ELISA of purified scFv reveal the binding of B1 and D7 to recombinant wtTDP-43.Wells of 96-well plate coated with wtTDP-43 diluted to 1 µg/mL were incubated with the control scFv, B1, or D7 at increasing concentrations of 1 µg/mL, 5 µg/mL, and 10 µg/mL (x-axis). The control scFv did not show signs of binding to TDP-43 at any concentration, while B1 and D7 showed binding via increasing absorbances. Mann Whitney test: **p = 0.004 or *p = 0.03 versus 5 µg of control scFv; ##p < 0.005 versus 10 µg of control scFv. N = 3-4.
Fig 3: B1 and D7 have no significant effect on total and soluble TDP-43 or its CTFs.Immunoblots of TDP-43 in the total (A) and soluble (C) protein lysates of HEK293T cells. Quantification of TDP-43 forms in the total (B) and soluble (D) lysates normalized to the TDP-43 condition (dotted line). Mock: empty vector. TDP-43 condition: cells transfected with TDP-43-expressing plasmid and empty vector. TDP-43/B1, TDP-43/D7 and TDP-43/control: TDP-43-expressing plasmid and intrabody-expressing plasmid. Mann-Whitney test, p > 0.5, (N = 3).
Fig 4: scFv D7 mediates the proteasomal degradation of the insoluble TDP-35 CTF.Immunoblots of TDP-43 in the insoluble protein fraction of HEK293T cells overexpressing TDP-43 and the intrabody in the absence (A) and presence (C) of autophagy and proteasome inhibitors. Quantification of the insoluble TDP-35 CTF normalized to the TDP43 condition (dotted line) in the absence (B) and presence (D) of autophagy and proteasome inhibitors. CHX: cycloheximide (10 µg/mL), Bafilomycin A1 (300 nM), MG-132 (0.5 µM). *p = 0.0286 vs TDP43; #p = 0.0286 vs TDP43/D7; Mann-Whitney test (N = 4). E) Immunoblot of TDP-43 in the insoluble protein fraction of HEK293T cells overexpressing TDP-43 and treated with autophagy or proteasome inhibitors. F) Quantification of insoluble TDP-43 forms normalized to the vehicle condition (dotted line). *p = 0.0286, Mann-Whitney test (N = 4).
Fig 5: TDP-43 modeling and in silico epitope mapping.A) 3D model of full-length TDP-43. B) Templates used for modeling the relative orientations of the different domains of TDP-43. C). View of the top 30 ranked predicted conformations for the complex between B1 and TDP-43 (i), and D7 and TDP-43 (ii). TDP-43 is shown in grey and the scFv in colors. D) In silico prediction of the interacting regions of TDP-43 with the scFvs B1 and D7.
Supplier Page from Abcam for Recombinant Human TDP43 protein (Tag Free)