α synuclein h3c

Fly heads were homogenized in 2× Laemmli buffer, boiled for 10 min, and centrifuged. SDS-PAGE was performed (Lonza, Basel, Switzerland) followed by transfer to nitrocellulose membrane (Bio-Rad, Hercules, CA) and microwave antigen retrieval in PBS. Membranes were blocked in 2% milk in PBS with 0.05% Tween-20 for 1 hr, then immunoblotted with appropriate primary antibody in 2% milk in PBS with 0.05% Tween-20 overnight at 4°C. Primary antibodies used include α-synuclein H3C (1:10,000 to 1:100,000, mouse, Developmental Studies Hybridoma Bank), GAPDH (mouse, 1:25,000 to 1:100,000, Invitrogen), GFP N86/8 (mouse, 1:100, Neuromab, Davis, CA). Membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (1:50,000) in 2% milk in PBS with 0.05% Tween-20 for 3 hr. Signal was developed with enhanced chemiluminescence (Thermo Scientific, Waltham, CA). Anti-GAPDH was used to demonstrate equivalent protein loading.

Fly heads were dissected then homogenized in 2× Laemmli buffer, boiled for 10 min, and centrifuged. SDS-PAGE was performed (Lonza) followed by transfer to nitrocellulose membrane (Bio-Rad) and microwave antigen retrieval in PBS. Membranes were blocked in 2% milk in PBS with 0.05% Tween-20 for 1 h, then immunoblotted with appropriate primary antibody in 2% milk in PBS with 0.05% Tween-20 overnight at 4 °C. Primary antibodies used include α-synuclein H3C (1:10,000 to 1:100,000, mouse, Developmental Studies Hybridoma Bank), α-synuclein clone 42 (1:5000 mouse, BD Bioscience), and phospho-serine 129 α-synuclein (1:5000, rabbit, Abcam). Membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (1:50,000) in 2% milk in PBS with 0.05% Tween-20 for 3 h. Signal was developed with enhanced chemiluminescence (Thermo Scientific).