Hrp conjugated goat anti mouse or anti rabbit igg antibodies

Samples (5 μg) were separated on 4–20% SDS polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked for 1 h at room temperature and probed with antibodies. HRP-conjugated goat anti-mouse or anti-rabbit IgG antibodies (Bio-Rad) were used as secondary antibodies. To examine the interaction of AAT with mitochondrial proteins, co-IP reactions were performed. Samples were washed with cold PBS and lysed in 0.5 mL IP buffer. Approximately 1 µg of AAT antibody was added to 45 µL of protein A Dynabeads. After 10 min incubation at room temperature, the beads were washed for 3 minutes (0.1% Triton X-100 in PBS). Lysates (200 μg of total cell lysate or 100 μg mouse mitochondria) were incubated with anti-AAT-conjugated beads overnight at 4 °C. The beads were then washed, suspended in gel loading buffer, and evaluated by Western blot. Signals were developed with ChemiDoc^TMTouch imaging system (Bio-Rad).

Samples were separated on 16% SDS polyacrylamide gel or pre-made 4–20% gradient SDS polyacrylamide gel (ThermoFisher) and transferred to PVDF membranes (GE Healthcare). The membranes were fixed with 4% paraformaldehyde and 0.1% glutaraldehyde for 30 min at room temperature and probed with various antibodies. The primary antibodies were anti-αSyn phospho (Ser129) (Abcam 51,253, 1:2500), anti-αSyn (BD Biosciences clone 42, 1:2500), anti-ATPIF1 (ThermoFisher, 1:1500), anti-ATP5A (Abcam, 1:1000), anti-GAPDH (Stressgen, 1:1000), anti-Calnexin (Sressgen, 1:1000), and anti-Syntaxin 6 (Sigma, 1:1000). The HRP-conjugated goat anti-mouse or anti-rabbit IgG antibodies (Bio-Rad) were used as the secondary antibodies. Signals were developed by ECL 2 substrate (Pierce) and scanned with ChemiDoc (Bio-Rad). The signal intensity was measured with the FIJI software.