Isolation of cytoplasmic proteins and subsequent western blot analysis

Neuronal cultures were harvested and centrifuged at 200 × g for 15 min to obtain cell pellets. Cytoplasmic fractions were isolated using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) following the manufacturer's instructions, but with minor modifications. Briefly, cells were resuspended in cytoplasmic extraction reagent-I and incubated for 10 minutes, followed by addition of cytoplasmic extraction reagent-II and centrifugation at 16,000×g for 5 minutes. Supernatants were considered the cytoplasmic fraction. The whole process was done on ice or at 4°C. The bicinchoninic acid (BCA) assay was used for protein quantitation. The same amount of proteins (30 μg) from each sample was mixed with Tricine-SDS sample buffer (Invitrogen) and 2% β-mercaptoethanol, boiled for 5 min and resolved by SDS-PAGE using 10–20% Tris/Tricine gel (Bio-Rad). Precision-plus protein standards (Bio-Rad) were included as references. After gel electrophoresis, proteins were transferred onto polyvinylidene difluoride (PVDF) membranes for further probing with antibodies against αS (Syn1, 610787, BD Biosciences) or αS (NACP98, Mayo Clinic [7]), H1.2 (ab17677, Abcam), H3 (ab1791, Abcam). Western Lightning Plus ECL (PerkinElmer) or ECL™ Prime Western Blotting Detection Reagent (Fisher Scientific) were used for visualization of protein immunoreactivities. Data from at least 3 sets of independent experiments were analyzed by one-way ANOVA with Dunnett’s post hoc test for statistical significance.