Fig. 2

Analysis of seeding efficiency of PSP-tau on Phospho-Plus tau variants. a. Schematic depiction of Phospho-Plus Ser/Thr → Glu tau variants generated on human 0N/4R P301L mutant tau. All numbers correspond to 2N/4R tau. R1-R4 depict microtubule-binding repeat regions. b–e. Representative immunoblot of the Complete Phospho-Plus (CPP) construct with all 19 sites mutated to Glu and seeded with Patients A-F shown. P301L tau seeded with Patient F or left unseeded are also shown. Total tau (t-tau) and AT8-tau (p-tau) shown from detergent-soluble (b, d) or insoluble (c, e) cell lysates. f–u. PSP-tau ( +) or unseeded (−) HEK293T cells transfected with different tau variants were fractionated into detergent-soluble and detergent-insoluble lysates and probed for total tau (f-g, t-tau) or AT8 (n–o, p-tau). Additional replicate blots are shown in Fig. S6. Quantitation of % aggregation (insoluble/[soluble + insoluble]*100) for each variant are shown (h-m). AT8 levels in detergent insoluble lysates has been normalized and shown (p-u). GAPDH is the loading control for detergent-soluble fraction. Broken lines (h–m) depict statistical tests done separately within groups of seeded or non-seeded tau variants. Numbers 1–5 denote the tau Variants; ‘P’, P301L tau; ‘G’, GFP; ‘ND’, not detected. Representative blots for all other patients not depicted here (A, C, D, E and F) are shown in Additional file 1: Fig. S6. Relative molecular masses (kDa) are indicated on the left of each blot. N = 3 experimental replicates. 1-way ANOVA with Dunnett’s test; ****P < 0.0001; **p < 0.01, *p < 0.05