Fig. 2

A Conformational stability assay (CSA) of sarkosyl-insoluble tau from AD brains applied as inoculum for neuronal cultures. Conformational profiles of human tau with incremental guanidine hydrochloride (Gdn HCl) concentrations to progressively unfold the misfolded tau to access epitope for interaction with 77G7 tau antibody (epitope 316–355 aa). AD-tau samples were a non-treated and b treated with Proteinase K before CSA, c the values of proteinase-resistant AD-tau were subtracted from non-treated AD-tau values. d Schematic representation of CSA. B Confocal images of primary neurons that we applied as the cell model. At 21DIV, primary neurons with distinguishable somatodendritic compartments (MAP2-positive, magenta) and axonal projections (Tau-positive, cyan). Mature primary neurons have functional synapses composed of pre- (Bassoon, green) and post-synaptic (SHANK3, red) compartments. Scale bars: 40 µm, cropped images: 10 µm. C The evolution of tau isoforms in primary cortical neuronal cultures: a the percentage of 3-repeat (3R) and 4-repeat (4R) tau isoforms in cell lysis of mouse cortical primary neurons in different days of cultures measured by conformation-dependent immunoassay (CDI) in three independent experiments. b Western blot of cell lysis of primary neurons in different days of cultures developed with 3R-specific tau antibody (clone: 2A1-1F4). D The amino acid sequence of mouse and human tau (2N4R isoform) varies in the N-terminal part of tau molecule, which is used in the production of mouse- and human-specific tau antibodies. Microtubule-binding domain composed of repeat domains (R1-R4) contains the pro-aggregation part of tau called paired helical filament (PHF) core and is identical in mouse and human tau