Samples were separated on Native-PAGE™ Bis-Tris Gels (Thermo Fisher Scientific) and transferred to PVDF membranes (Bio-Rad). The membranes were blocked with 5% BSA in TBST (Tris-buffered Saline-Tween 20) overnight at 4 °C. Then the membranes were incubated with purified MBP-PFFNB2-FLAG (~4 μg/ml) or mouse anti-α-syn mAb (1:2000 dilution, BD Biosciences, Cat no. 610787) in TBST with 5% Bovine Serum Albumin overnight at 4 °C. Mouse-anti-Flag-HRP antibody (1:5000 dilution, Sigma-Aldrich, Cat no. A8592) or anti-mouse IgG-HRP (1:5000 dilution, GE Healthcare, Cat no. NA931) were used as secondary antibody followed by incubation with SuperSignal West Pico Plus chemiluminescent substrate (Thermo Fisher Scientific). The images were acquired and processed with ImageQuant LAS 4000mini scanner (GE Healthcare Life Sciences) and Amersham Image 600 (GE Healthcare Life Sciences).
The soluble and insoluble fractions of brain lysates were resolved on 15% Tris-glycine gel and transferred to PVDF membranes for analysis with rabbit anti-α-syn (1:1000 dilution, Cell signaling, Cat#4179), rabbit anti-α-syn (pS129) (dilution 1:1000, Abcam, Cat no. ab51253) and mouse anti-ß-actin peroxidase (1:10,000 dilution, Millipore Sigma, Cat no. A3854) antibodies.
The soluble and insoluble fractions of brain lysates were resolved on 15% Tris-glycine gel and transferred to PVDF membranes for analysis with rabbit anti-α-syn (1:1000 dilution, Cell signaling, Cat#4179), rabbit anti-α-syn (pS129) (dilution 1:1000, Abcam, Cat no. ab51253) and mouse anti-ß-actin peroxidase (1:10,000 dilution, Millipore Sigma, Cat no. A3854) antibodies.