Ab51252

After separation of protein samples on SDS-PAGE, proteins were transferred to 0.45 µm Immobilon-PVDF membranes (Millipore) pre-activated with methanol in standard Tris-Glycine transfer buffer (pH 8.3) supplemented with 20% methanol for 2 h at 400 mA. Membranes were blocked in 5% skim milk powder in TBS-T for 1 h, followed by incubation with appropriate primary antibodies on an orbital shaker at 4 °C overnight. Membranes were rinsed thoroughly in TBS-T, followed by incubation with appropriate secondary HRP-conjugated antibodies for 1 h on an orbital shaker at room temperature. Membranes were rinsed thoroughly and developed using WesternBright ECL (Advansta) in a MyECL Imager v 2.2.0.1250 (Thermo Scientific) according to the manufacturer’s instructions.
Primary antibodies used for Western blots include anti-HA(R) rabbit (1:6000, ab9110, Abcam), anti-HA(M) mouse (1:1000, ab18181, Abcam), anti-PSMD1 (1:1000, ab2941, Abcam), anti-PSMD2 (1:1000, PAB6715, Abnova), anti-PSMA3 (1:500, sc-58417, Santa Cruz), anti-PSMA1 (1:1000, ab140499, Abcam), anti-FLAG (1:2500, F3165 Clone M2, Sigma), anti-GFP (1:2500, ab290, Abcam), anti-p53 HRP (1:2500, HAF1355, Biotest), anti-α-synuclein (1:500, ab51252, Abcam), anti-GAPDH (1:1000, MAB374, Clone 6C5, Millipore), anti-CBR3 (1:1000, sc-374393, Santa Cruz).

Immunofluorescence images of fixed cells electroporated with reduced or oxidized α-Syn were acquired on a Zeiss LSM ultraviolet confocal microscope. After electroporation, cells were recovered for 5 h at 37 °C, 5% CO₂ on poly-L-lysine-coated 25-mm cover slips. Slides were washed with PBS and fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton-X/PBS for 3 min. After 3 × 10 min PBS washes, samples were blocked with 0.13% gelatin from cold-water fish skin (Sigma-Aldrich) for 1 h. Slides were incubated for 2 h in 0.13% gelatin with anti-α-Syn antibody (1:500 Abcam ab51252), washed 3 × 10 min with PBS and incubated with anti-Rabbit IgG Atto647 (40839 Sigma-Aldrich, 1:1,000 dilution) for 1.5 h in 0.13% gelatin. Cells were washed 3 × 10 min with PBS and stained with 2 μg ml⁻¹ 4,6-diamidino-2-phenylindole (Invitrogen) in PBS for 15 min. Images were acquired at × 60 magnification.