Figure 3.

Pharmacologic stabilization of ^FTTTR^A25T tetramers partially restores secretion in native conformations. (A) Graph showing total (filled bars) and tetrameric (open bars) ^FTTTR^A25T in conditioned media prepared in the presence of Taf-S or Taf on HEK293T cells treated for 16 h with Tg (500 nM) and/or ISRIB (200 nM). Total and tetrameric ^FTTTR^A25T are normalized to media prepared on vehicle-treated cells in the presence of Taf-S. Error bars show SEM for n = 8 independent experiments. (B) Representative chromatogram showing ^FTTTR^A25T tetramers in conditioned media prepared in the presence of Taf-S (left) or Taf (right) on HEK293T cells treated for 16 h with Tg (500 nM) and/or ISRIB (200 nM). ^FTTTR^A25T tetramers were separated on anion exchange chromatography using the UPLC system and visualized by compound 1 fluorescence. (C) Representative CN-PAGE/immunoblot (left) and quantification (right) of ^FTTTR^A25T aggregates in conditioned media prepared in the presence of Taf-S or Taf on HEK293T cells treated for 16 h with Tg (500 nM) and/or ISRIB (200 nM). Total ^FTTTR^A25T in each condition is shown in the SDS-PAGE immunoblot. Error bars show SEM for n = 5 independent experiments. (D) Graph showing total (filled bars) and tetrameric (open bars) ^FTTTR^A25T in conditioned media prepared in the presence of Taf-S or Taf on HEK293T cells treated for 16 h with Tg (500 nM) and/or ISRIB (200 nM). Total and tetrameric ^FTTTR^A25T are normalized to media prepared on vehicle-treated cells in the presence of Taf. Relative populations of non-native TTR in each condition are shown. Error bars show SEM for n = 8 independent experiments. *Indicates p < 0.05, **p < 0.01, and ***p < 0.005 for a two-tailed paired t-test.