Optima max ultracentrifuge

The kinetics of amyloid formation was followed using a 96-well format Thioflavin T (ThT) binding assay. Cleared protein stocks (0.2 mM protein in 5 mM HEPES, pH 7.5, and 0.1 mM CaCl₂) were diluted with distinct buffers to 14–150 µM protein and supplemented with 10 μM ThT. The buffers for the reactions were 25 mM Tris-HCl, pH 7.5, 0.1 M NaCl containing 4 mM either EDTA or CaCl₂ (control for no aggregation) and 50 mM Gly, pH 1.6. Assays were initiated by placing the sealed 96-well plate at 37 °C in a POLARstar (BMG Labtech, Germany) microplate reader. The ThT fluorescence was measured through the bottom of the plate every 30 min with a 450 nm excitation filter and a 480 nm emission filter. All measurements were performed in duplicate, and the experiments were repeated twice with different protein batches. When required, aggregates were harvested from the reaction mixtures by a 100,000 x g centrifugation for 1 h using an Optima Tm^Max ultracentrifuge (Beckman, USA). The resulting pellet and supernatant fractions were used for protein concentration determination. Before use, pellet suspensions were sonicated in a bath sonicator for 5 min.

The MelB_St reconstitution was performed as described previously²⁸ . Briefly, 5.6 mg of E. coli polar lipids extract at a concentration of 40 mg/mL solubilized in 7.5% DDM was added to 1 mL of 1 mg of MelB_St in 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10% glycerol, and 0.35% UDM (~1:350 mole/mole), and incubated for 10 min on ice. MSP1E3D1 proteins were then added into the MelB_St/lipid mixture at a 5:1 mole ratio (MSP1E3D1:MelB_St), and incubated at room temperature with mild stirring for 30 min before being shifted to 4 °C with mild stirring. The detergents were removed and subsequent MelB_St lipids nanodiscs were formed by stepwise additions of Bio-beads SM-2 resin (500 mg and then an additional 300 mg after 2 h) and further incubated overnight. After separating the Bio-beads SM-2 resins, the MelB_St lipids nanodiscs were isolated by absorbing onto Ni-NTA beads and the MelB_St-free empty nanodiscs were in the void due to His-tag being removed. The MelB_St lipids nanodiscs were eluted by adding 250 mM imidazole and the elutes were dialyzed against 20 mM Tris-HCl, pH 7.5, and 150 mM NaCl, and concentrated to ~ 3–4 mg/mL. After ultracentrifugation at ~352000 g (90,000 rpm using a TLA-120 rotor) in a Beckman OptimaTM MAX Ultracentrifuge for 30 min 4 °C, the supernatant containing MelB_St lipids nanodiscs were stored at −80 °C after flash-frozen with liquid nitrogen.

To identify IFT172 in subcellular compartments, RPE-1 cells (ATCC) were fractionated³⁵ . Cells from two 10 cm cell culture plates were collected in 4 ml lysis buffer (25 mM Tris-HCl, pH 7.5, 50 mM sucrose, 0.5 mM MgCl₂, 0.2 mM EGTA), followed by homogenization with a Dounce homogenizer. The sucrose concentration was restored to 250 mM afterwards. Nuclear materials were removed by centrifugation at 1000 × g for 10 min (Multifuge 1 L, Heraeus). The supernatant was collected and further centrifuged at 195,000 xg for 30 min (Optima MAX Ultracentrifuge, Beckman Coulter) to separate cytosolic (supernatant) and organellar (pellet) fractions. The pellet was resuspended in SDS buffer (2.5% SDS, 50 mM Tris pH 8.1) for western blotting. Total cell lysate, cytosolic fraction and organellar fraction were separated by SDS-PAGE and analyzed with Western blot analysis. Anti-IFT172 antibody used with 1:100 dilution and goat anti-mouse IgG (62-6520, Thermo Fisher) was used as secondary antibody with 1:5000 dilution. Uncropped blots are available in Supplementary Figure 5.

Following ITC analysis, the solutions containing HIV protein and RNA complex was spun at 100,000 g for 1 hr (TLA 100.2 rotor; optima max ultracentrifuge; Beckman). The supernatant was removed and the pellet was resuspended in 50μl of TBS. Protein estimation was done using UV-Vis (A280) on both the supernatant and the pelletable materials (NanoDrop1000; Thermo Scientific) via Bradford Protein Assay [47 (link)].

HTT and chaperone proteins were thawed on ice and transferred to centrifugation tubes (Microfuge Tube Polypropylene, Beckman Coulter). Ultracentrifugation (Optima MAX Ultracentrifuge, Beckman) was carried out at 50000 rpm for 1 h at 4 °C. Protein supernatants (around 95% of the initial volume) were transferred to low-binding tubes, and molar concentration was determined via Bradford assay. Samples containing 5–10 μM total protein and 0.5–1 mM SDA were incubated for 30 min at room temperature. Cross-linking was stopped by adding 1 M Tris-base, samples were then put on ice and photoactivated for 30 min under UV light. SDA cross-linked proteins were loaded on SDS-PAGE and subjected to in-gel digestion. In brief, gel bands were reduced with 5 mM DTT at 56 °C for 30 min and alkylated with 40 mM chloroacetamide at room temperature for 30 min in the dark. Protein digestion was carried out using elastase at an enzyme-to-protein ratio of 1:20 (w/w) at 37 °C overnight.