Figure 3. Stress-Independent ATF6 Pre-activation Increases Partitioning of Destabilized TTR Monomers to ER Degradation Pathways.

(A) Bar graph comparing the fraction remaining of [³⁵S]-labeled ^FTTTR variants at 4 hr in the absence (gray) or presence (blue) of ATF6 preactivation. Fraction remaining was calculated from autoradiograms as shown in Figure S1A, using the equation: Fraction Remaining = {Total ^FTTTR Signal in Lysate at Time = t } + {Total ^FTTTR Signal in Media at Time = t } divided by {Total ^FTTTR Signal in Lysate at Time = 0 } + {Total ^FTTTR Signal in Media at Time = 0 } (Sekijima et al., 2005; Shoulders et al., 2013). Shading shows familial disease-associated ^FTTTR variants (pink) and ^FTTTR variants not involved in familial disease (brown). Error bars represent SEM from biological replicates (n ≥ 3).

(B) Plot relating the impact of ATF6 activation on the fraction remaining for ^FTTTR variants, as shown in Figure 3A, to each respective TTR variants’ CSS (Table S1). The impact of ATF6 activation on ^FTTTR variant fraction remaining is defined as {fraction ^FTTTR remaining t = 4 hr in the presence of ATF6} divided by {fraction ^FTTTR remaining t = 4 hr in the absence of ATF6}. Error bars represent SEM from biological replicates (n ≥ 3).

(C) Quantification of fraction remaining for [³⁵S]-labeled ^FTTTR^D18G in HEK293^DAX following ATF6 preactivation (10 μM TMP; 16 hr) when chased in the presence of vehicle (black), MG132 (10 μM; orange), NH₄Cl (25 mM; green), or both MG132 and NH₄Cl (purple). A representative autoradiogram and experimental protocol employed are shown in Figure S2A. Error bars represent SEM from biological replicates (n ≥ 3).

(D) Quantification of fraction remaining of [³⁵S]-labeled ^FTM-TTR in HEK293^DAX in the absence (black) or presence (blue) of ATF6 preactivation (10 μM TMP; 16 hr). A representative autoradiogram is shown in Figure S2D. Error bars represent SEM from biological replicates (n = 6).

*p < 0.05, **p < 0.01, ***p < 0.001.