Immobilon luminata forte enhanced chemiluminescence

Culture media was conditioned for last 24 h of experiment. Media was removed from cells and centrifuged at 1000 × g for 5 min to remove floating cells. Cells were harvested and lysed in 1% (v/v) TX-100 in PBS and total protein content of well calculated using the BCA assay. Media was applied to PROTRAN nitrocellulose (Perkin Elmer) in triplicate using a dot blot apparatus (Bio-Rad). A PFF standard curve was also applied with serial dilution of PFF in culture media (0–30 ng/ml). Following two PBS washes, the membrane was blocked with BlockACE (BioRad) and incubated overnight at 4°C with anti-α-syn filament antibody (abcam, ab209538). Following incubation with anti-rabbit HRP-conjugated secondary antibody, membranes were incubated with Immobilon Luminata Forte enhanced chemiluminescence (Merck) and images acquired and quantified using Image Lab software (BioRad). Dot density was converted to ng/ml using the PFF standard curve and normalized to protein content of the well. The mean of each triplicate was calculated and expressed as ng fibrils/mg protein.

Protein was loaded on 4–12% Bis-Tris NuPAGE gels (ThermoFisher), separated by electrophoresis and transferred to Hybond PVDF membrane (GE Healthcare). For TX-100 soluble/insoluble studies, the amount of urea-SDS fraction loaded for each sample was calculated as a proportion of the protein concentration in the TX-100 soluble fraction. Because of the purity of the insoluble fraction neither β-actin nor GAPDH were detectable in the insoluble fraction. For α-syn blots, PVDF was fixed with 4% paraformaldehyde and 0.01% (v/v) glutaraldehyde for 30 min at room temperature (62 (link)). Membranes were blocked with 5% milk/PBS/0.1% (v/v) Tween 20 and incubated with the following primary antibodies: α-syn (abcam, ab1903 for mouse; ab80627 for human), α-syn phospho S129 (abcam, ab51253), β-actin (abcam, ab6276), GAPDH, (abcam, ab8245), GCase (Merck, 2E2 for human; Sigma, G4171 for mouse), GRP78/BiP (abcam, ab21685), HA (Biolegend, HA.11), histone H3 (abcam, ab1791), LIMP2 (abcam, ab16522), TFEB (abcam, ab2636), TH (abcam, ab112). Following incubation with respective HRP-conjugated secondary antibodies, blots were incubated with Immobilon Luminata Forte enhanced chemiluminescence (Merck) and images acquired and quantified using Image Lab software (BioRad). Band densities were normalized to β-actin or GAPDH.