Fuji las 3000 intelligent dark box

Recombinant human 2N4R tau was resuspended in 2× kinase activity buffer (100 mM Hepes, pH 7.5 20 mM MgCl₂, 2 mM EGTA) and incubated for 1 h at 37°C with ATP (8:1 mixture of non‐labeled and (γ‐32)‐labeled ATP, 10 µM final concentration) alone or in a 50:1 ratio with recombinant human 62.2 kDa EIF2AK2 (PKR) protein (Thermo Fisher Scientific). The samples were subjected to SDS‐PAGE and stained at RT, using Coomassie Brilliant Blue, destained ON at RT and left for 1 h at RT in a 35% EtOH, 3.5% glycerol solution prior to drying. The dried gel was photographed on Fuji LAS‐3000 Intelligent Dark Box (Fujifilm, Japan) and developed using standard autoradiography.
For phosphorylation of recombinant human 2N4R tau to be subsequently analyzed by immunoblot analysis, in vitro phosphorylation was performed using pure, non‐labeled ATP and incubated 18 h at 37°C in 1× kinase activity buffer and subjected to immunoblotting as previously described for whole‐cell extracts.

Samples were spotted by non-denaturing immuno-dot blot as previously described [53 (link)]. Briefly, different amounts of PFF (3.12–100 ng), monomeric α-Syn (100 ng), or vehicle (PBS) were dotted directly onto a nitrocellulose membrane using a vacuum filtration system (Bio-Rad Bio-Dot Apparatus). Membranes were incubated with primary antibodies anti-α-Syn Syn-1 (BD Biosciences #610,787, 1:1000), anti-p25α (produced in house [58 (link)], affinity rabbit polyclonal, 1:1000), and aggregate-specific MJF14-6–4-2 (MJF14) (Abcam #209,538, 1:45,000) and fibril-specific FILA-1 (produced in house [59 (link)], 1:1,000). Proteins were visualised using ECL in a Fuji LAS-3000 Intelligent Dark Box (Fujifilm, Japan).

Lyophilized recombinant α-syn wt, α-syn T64A/T72A, and α-syn T64E/T72E was re-suspended in sterile PBS (Gibco) to a final concentration of 2 mg/mL and placed in an Eppendorf Thermotop shaker at 37 °C, 1050 rpm. To test for aggregation, a small fraction from each sample was withdrawn at day 0, 1, 4 and 10 and tested for the Thioflavin-T (ThT) signal. In brief, 100 µL of 40 µM ThT dissolved in measure buffer (90 mM glycine, pH 8.6) was added to 10 µL of each protein sample and incubated for 5 min at RT before measuring ThT signal on an EnSpire 2300 Multilabel Plate Reader (PerkinElmer Life Sciences) (excitation at 450 nm and emission at 486 nm). The signal was normalized to the ThT signal from 0 days of incubation.
For sedimentation, samples incubated for 10 days were centrifuged at 25,000 x g, 30 min at RT, and the supernatant was transferred to a new tube. The pellet was resuspended in an equal volume of PBS. Same volume of each sample was subjected to SDS-PAGE, stained at RT using Coomassie Brilliant Blue, destained ON at RT and left for 1 h at RT in a 35% EtOH, 3.5% glycerol solution prior to drying. The dried gel was photographed on Fuji LAS-3000 Intelligent Dark Box (Fujifilm, Japan).