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. 2019 Feb 7;294(15):6130–6141. doi: 10.1074/jbc.RA118.005257

Figure 5.

Figure 5.

TabFH2 inhibits amyloid seeding caused by ex vivo ATTR seeds extracted from all cardiac extracts studied. a, inhibition of amyloid seeding by TabFH2 in the presence of cardiac ex vivo seeds, measured by protein content quantification of insoluble fractions. 30 ng/μl of ex vivo seeds extracted from the hearts of eight ATTR patients were added to 0.5 mg/ml of recombinant WT transthyretin in the presence of 0, 180, or 360 μm TabFH2. The insoluble fractions were collected by centrifugation after 24 h of incubation. The insoluble protein content was measured by absorbance at 280 nm. The results show that full inhibition of amyloid seeding by TabFH2 is achieved at the highest concentration for every ATTR ex vivo sample. n = 3. Error bars, S.D. *, p ≤ 0.05; **, p ≤ 0.005; ***, p ≤ 0.0005. b, TTR immunodot blot of insoluble fractions collected by centrifugation from the assay shown in a, with consistent results. The experiment shown in a and b was performed in combination with the assay shown in Fig. 2; therefore the control samples without seeds are the same. All samples, including those from Fig. 2, were spotted onto the same nitrocellulose membrane and subjected to the same procedure; splicing was needed for presentation purposes. c, electron micrographs of the samples containing 360 μm TabFH2 collected from a. These are uncorrected images generated directly from a Gatan 2kX2k CCD camera. Scale bar, 200 nm. d, correlation between amyloid seeding capacity in the presence of 180 μm TabFH2, and relative quantity of truncated TTR of ex vivo ATTR seeds. Notice that the presence of truncated TTR facilitates seeding. Truncated TTR content was quantified by ImageJ from two independent Western blots. Lineal regression and Pearson r were obtained by OriginLab. e, comparison of inhibition of ATTR-V30M amyloid seeding by tafamidis, diflunisal, and TabFH2, measured by ThT fluorescence. 180 or 360 μm inhibitor was added to 0.5 mg/ml of recombinant WT TTR and 30 ng/μl of ATTR-V30M seeds. All replicates are shown, n = 4. a.u., arbitrary units. Inset, anti-TTR dot-blot of insoluble fractions collected by centrifugation after 24 h of incubation. f, electron micrographs of the samples collected from e.