Ir dye 680 or 800 antibodies

Proteins were resolved on 4%–20% Criterion TGX pre-cast gels (Biorad) and transferred to membranes by semi-dry trans-Blot Turbo transfer system (Biorad). The membranes were blocked with Odyssey Blocking Buffer (Li-Cor Cat #927–40000) and then incubated for 1 hour at RT or overnight at 4°C with the indicated primary antibody. The membranes were washed in TBST (3 × 5 min) followed by incubation for 1 hour at RT with fluorescently conjugated goat anti-mouse or rabbit IR Dye 680 or 800 antibodies (LICOR). The blots were washed in TBST (3 × 5 min) and scanned on an ODYSSEY® CLx (Li-Cor). Quantitation of western blots was performed using Image Studio (Li-Cor).

Proteins were extracted in cell lysis buffer (Cell Signaling Technology) with additional phosphatase and protease inhibitors (Sigma). Proteins were separated on 4–20% Criterion TGX pre-cast gels (Biorad) in SDS/Tris-glycine running buffer and transferred to PVDF membranes by semi-dry trans-Blot Turbo transfer system (Biorad). The membranes were blocked for 1h with Odyssey Blocking Buffer (Licor) and then incubated for 1h at room temperature (RT) with antibodies: anti α-syn (Cell Signaling Technologies, 1:1000, rabbit), anti-VDAC1 (Abeam, 1:2000, mouse), anti-GAPDH (Sigma, 1:2000, rabbit), and anti-β-actin (Santa Cruz, 1:1000, mouse). The membranes were washed in TBST (3×5 min) at RT followed by incubation for 1h at RT with fluorescently conjugated goat anti-mouse or rabbit IR Dye 680 or 800 antibodies (Licor). The blots were washed in TBST (3×5 min) at RT and scanned on an ODYSSEY^® CLx (Licor). Quantitation of western blots was performed using Image Studio (Licor), the intensity of target proteins was standardized with the loading control.