S5136

The ability of test compounds to prevent TTR aggregation was evaluated under the acidic conditions that favor TTR aggregation and fibril formation. A 2 μL solution of 167 μM human TTR (ACRO Biosystems #H5223) was incubated with 7 μL of 50 mM sodium acetate pH 4.0 (Sigma-Aldrich #S7545) and 100 mM KCl (Sigma-Aldrich #S5405) in the presence or absence of 1 μL TTR inhibitor for 72 h at 37 °C. At the end of the incubation, 3.5 μL of 500 mM sodium phosphate (Sigma-Aldrich #S5136) buffer pH = 8.0 was added to each sample for neutralization and 0.6 μL of 5% CHAPS (Sigma-Aldrich #C5070) as a detergent to prevent reassociation of protein. The cross-linking was performed by adding 1.5 μL of 5% glutaraldehyde solution (Sigma-Aldrich #G6257). After 4 min, the reaction was stopped by the addition of 2.5 μL freshly made 5% NaBH₄. The samples were subjected to TTR Western blotting with prealbumin antibodies (1:500; Dako #A0002). Band intensity for TTR monomer and TTR aggregates was quantified from scanned images of the blots.

The ability of test compounds to prevent TTR aggregation was evaluated under the acidic conditions that favor TTR aggregation and fibril formation.^{63 (link),64 (link)} A 2 μL solution of 167 μM human TTR (ACRO Biosystems #H5223) was incubated with 7 μL of 50 mM sodium acetate (pH = 4.0) (Sigma-Aldrich # S7545), and 100 mM KCl (Sigma-Aldrich # S5405) in the presence or absence of 1 μL of TTR inhibitor for 72 h at 37 °C. At the end of the incubation, 3.5 μL of a 500 mM sodium phosphate (Sigma-Aldrich #S5136) buffer (pH = 8.0) was added to each sample for neutralization and 0.6 μL of 5% CHAPS (Sigma-Aldrich #C5070) as a detergent to prevent reassociation of protein. The cross-linking was performed by adding 1.5 μL of 5% glutaraldehyde solution (Sigma-Aldrich # G6257). After 4 min, the reaction was stopped by the addition of 2.5 μL of freshly made 5% sodium borohydride (NaBH₄). Samples were subjected to TTR Western blotting with prealbumin antibodies (1:500; Dako #A0002). Band intensity for TTR monomer and TTR aggregates was quantified from scanned images of the blots.