Ab97370

Cells were lysed in RIPA and 1:100 protease inhibitor (Sigma-Aldrich). After centrifugation, the supernatant was collected, and protein content was evaluated by the BCA protein assay kit (Thermo Fisher 23225). 80 µg of each sample under reducing conditions were loaded into each lane and separated by electrophoresis on a 10% SDS polyacrylamide gel. Following electrophoresis, proteins were transferred to Immobilon transfer membranes (Tamar). Non-specific binding was blocked by incubation with 5% milk in TBST (10 mM Tris–HCl (pH 8.0), 150 mM NaCl, 0.1% Tween 20) for 1 h at 25 °C. Membranes were subsequently incubated with antibodies against ASL (1:500, ab97370, Abcam), p97 (1:10,000, PA5-22257, Thermo Scientific), GAPDH (1:1,0000, ab128915, Abcam), TH (1:500, CST-2792S, cell signaling) α-synuclein. Antibody was detected using peroxidase-conjugated AffiniPure goat anti-rabbit IgG or goat anti-mouse IgG (Jackson ImmunoResearch) and enhanced using chemiluminescence western blotting detection reagents (Pierce™ ECL Western Blotting Substrate, Thermo Fisher). Gels were quantified by Gel Doc XR + (BioRad) and analyzed by ImageLab 6.0 software (BioRad). The band area was calculated by the band's intensity divided by the value obtained from the loading control.