Gel doc 1000 uv

SNc protein was extracted (n = 6 mice per group), separated using 12–15% SDS–PAGE, and transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk for 1.5 hours at room temperature and incubated with antibodies against TH, α-synuclein, p62, Beclin 1, LC3, p-AKT, AKT, p-mTOR, mTOR, and GAPDH at 4° C overnight. The next day, membranes were rinsed with tris-buffered saline containing 0.1% Tween 20 and hybridized with corresponding secondary antibodies for 1.5 hours. Proteins were visualized using enhanced chemiluminescence, and immunoreactive bands were analyzed with image analysis software (Gel Doc 1000-UV; Bio-Rad, Richmond, CA) to calculate the optical density of α-synuclein and TH bands normalized to GADPH.
Information of antibodies: rabbit polyclonal LC3 antibody: 1:1000. Rabbit polyclonal P62 antibody: 1:1000. Beclin 1 polyclonal antibody: 1:1000. Mice anti TH monoclonal antibody: 1:1000. Rabbit polyclonal α-synuclein antibody: 1:1000.

The SNpc protein was extracted (n = 6 mice per group), separated using 12–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% nonfat milk for 1.5 h at room temperature and incubated with antibodies against TH, α-synuclein, and GAPDH at 4°C overnight. The next day, membranes were rinsed with tris-buffered saline containing 0.1% Tween^® 20 and hybridized with HRP-conjugated anti-rabbit immunoglobulin G for 1.5 h. Proteins were visualized using enhanced chemiluminescence, and immunoreactive bands were analyzed with image analysis software (Gel Doc 1000-UV; Bio-Rad, Richmond, CA) to calculate the optical density of α-synuclein and TH bands normalized to GADPH.