Fig. 4

Inhibition of in-vitro aggregation of pS422-tau by dmCBTAU-22.1. a Schematic representation of the chemical ligation process applied to preparation pS422-tau. Ser⁴²² (red) is phosphorylated in the CBTAU-22.1 epitope and Ser⁴¹⁶ (blue) is mutated into Cys as a result of the chemical ligation process. b SDS-PAGE analysis of the ligation reaction progress. Lane 1 corresponds to the full tau1–415-Mxe-CBD multidomain protein produced in E. coli after cloning into the pTXB1 vector. For the purposes of this analysis the protein was purified via His-tag affinity chromatography. Lane 2 corresponds to the thioester product obtained by treatment of the Lane 1 product with excess MESNa. Lane 3 represents the ligation product obtained by reaction of the Lane 2 product with tau peptide CL22D-P. c Size Exclusion Chromatography profiles of pS422-tau (green) and non-phosphorylated 2N4R-tau (blue). d Western blot of pS422-tau and non-phosphorylated 2N4R-tau with dmCBTAU-22.1. e Aggregation of pS422-tau in the absence (black) or presence of dmCBTAU-27.1 (red) or dmCBTAU-22.1 (blue) as monitored continuously by ThT fluorescence. The molar ratio between pS422-tau and IgG was 1: 0.6 in both cases. Each experimental condition was tested in three independent replicates, the red triplicates and two of the blue triplicates overlap and cannot be visually distinguished. f Atomic force microscopy of pS422-tau fibrils. The two panels correspond to AFM images with sizes of 6 × 6 μm and 1.5 × 1.5 μm, respectively