Epr5526

Filter retardation assay (FRA) was performed as previously described (Ast et al., 2018b (link)), utilizing a final protein concentration of 1.5 μM. The addition of PP started the aggregation process. At the indicated time points at 0, 4, 8 and 24 h after addition of PP, an aliquot was taken from each sample and flash frozen in liquid nitrogen. Before blotting, samples were mixed 1:1 v/v with a stop solution (4% SDS, 100 mM DTT). In a dot blot apparatus attached to a pump, whole samples were loaded on and filtered simultaneously through a cellulose acetate membrane with 0.2 μm pores (OE66, GE Healthcare, #10404180) pre-soaked in TBS buffer containing 1% SDS. All blot wells were washed twice with TBS plus 0.1% SDS and membranes were then blocked in 5% skim milk in TBS-T. Fibrils retained on the membrane were detected by probing with both anti-HTTEx1 primary antibodies EPR5526 (1:1000; Abcam) and MW8 (1:2000; DSHB). Anti-rabbit HRP secondary antibody (1:10,000; #31460, Thermo Fisher) was used to probe HTTEx1 EPR5526 and IRDye680 anti-mouse (1:10.000; #926-68070 LI-COR Biosciences) was used for detection of MW8. HRP signals were developed with Pierce ECL Western Blotting Substrate (#32209, Thermo Fisher) and imaged using the OdysseyFC imaging system (LI-COR Biosciences).

Tissue samples were homogenized in liquid nitrogen using a mortar and lyzed in RIPA buffer (150 mM NaCl, 5 mM EDTA pH 8, 0.05% NP-40, 1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 50 mM Tris-HCl pH 7.4, inhibitors of phosphatases and proteases), sonicated for 15 min, and centrifuged at 20,000 g, for 15 min, at 4°C. Samples (10 µg of total protein) were loaded onto 3-8% or 4-12% Tris-acetate gel (#EA03758, LifeTech) and run at 150 V. Gel was transferred onto nitrocellulose membrane, blocked in 5% skimmed milk and probed overnight with anti-HTT antibody diluted in 5% milk (EPR5526, Abcam, 1:3000) at 4°C. Memcode protein staining (LifeTech) was used for normalization of loading. Secondary antibody conjugated with HRP (anti-mouse, #711-035-152, Jackson ImmunoResearch, 1:10,000 or anti-rabbit, #711-035-152, Jackson ImmunoResearch, 1:10,000) was used. The signal was revealed by chemiluminiscence (ECL, #28980926, APCzech) and detected by The ChemiDoc XRS+system (Bio-Rad).