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. Author manuscript; available in PMC: 2017 Nov 1.
Published in final edited form as: Neurobiol Aging. 2016 Jul 29;47:113–126. doi: 10.1016/j.neurobiolaging.2016.07.015

Fig. 3.

Fig. 3

Biochemical analysis of PAD exposure and oligomerization for all tau isoforms. (A and B) Sandwich ELISAs were used as non-denaturing assays to capture PAD exposed tau (A, TNT1 capture antibody) and tau oligomers (B, TOC1 capture antibody), with detection of captured tau using the rabbit pan-tau antibody, R1. (A) The TNT1 ELISAs show significantly higher signal in aggregated samples compared to monomeric samples for each tau isoform. TNT1 signal was highest in hT24 aggregates, which reached significance compared to hT40, hT39, hT37 and hT23 aggregates. hT40, hT34 and hT39 aggregates were significantly higher when compared to hT37 and hT23 aggregates. hT39 monomers were significantly different compared to hT34, hT24, hT37 and hT23 monomers. (B) Similarly, the TOC1 ELISAs show significantly higher signal in aggregated samples compared to monomeric samples for each tau isoform. TOC1 signal was highest in hT24 aggregates, which reached significance when compared to hT40, hT39, hT37 and hT23 aggregates. hT40 and hT39 aggregates were significantly greater than hT37 and hT23 aggregates, while hT34 aggregates were significantly higher when compared hT39, hT37 and hT23. hT39 monomers were significantly different compared to hT24, hT37 and hT23 monomers. The data were compared using the Kruskal-Wallis ANOVA with Dunn post-hoc (isoform monomers), oneway ANOVA with Holm-Sidak post-hoc (isoform aggregates) and the Mann-Whitney test (monomers vs aggregates). *p < 0.05 vs. the monomer of the same isoform; **p < 0.05 vs. hT37 and hT23 aggregates; ***p < 0.05 vs. hT40, hT39, hT37 and hT23 aggregates; #p < 0.05 vs. hT24 and hT23 monomers..(C–H) The same samples that were used for sandwich ELISAs were analyzed using SDS-PAGE/western blotting as a denaturing assay (5 ml of 2 mM sample loaded in each lane). In TNT1 and TOC1 blots, the monomer and aggregate samples produced equal signal (Tau5 was used to normalize TNT1 and TOC1 signals, Student’s t-test, all p > 0.05) because denaturation of the proteins exposes the epitopes making them equally accessible. Collectively, these data indicate that all tau isoforms have PAD exposed and form oligomers when induced to aggregate in vitro, and TNT1 and TOC1 strongly label aggregated forms of all tau isoforms, not monomers, in a conformation-dependent manner.