Triton-X 100 soluble/insoluble fractions

HEK cells were transiently transfected with empty vector or GFP-αSN/αSN-RFP as previously described. Cells were treated with either PBS or PFF seeds (4 μg/mL) 24 h after transfection. After 24 h of treatment, cells were harvested in cold lysis buffer (50 mM Tris/HCl pH 7.4, 150 mM NaCl, 5 mM EDTA pH 8.0) with protease and phosphatase inhibitors and 1% Triton-X 100 and incubated on ice for 30 min. The lysates were then centrifuged at 25,000 × g for 60 min at 4 °C. The supernatant was collected as the Triton-X 100 soluble fraction and the pellet was resuspended in lysis buffer with 2% SDS and sonicated for 10 s and collected as the Triton-X 100 insoluble fraction. Both fractions were boiled and then a BCA protein assay was used to quantify the protein concentration of the samples. Approximately 12 μg of total protein was loaded onto a 4–20% mini-Protean TGX gel (Bio-Rad, Hercules, CA), and blots were evaluated as described previously.