Source data Figure 5 Yuan et al_iScience_2025
Description
Full images of western blots used in Figure 5J-K from Yuan et al. Modulation of striatal cAMP levels: a key pathway in the treatment of hyperkinetic movement disorders ISCIENCE-D-25-11384R1
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Western Blot Mice were decapitated and their heads were instantly frozen for 12s in liquid nitrogen and stored at -80°C before use. Fur and skull were eliminated on dry ice and the frozen brains were cut into 210 µm sections using a cryostat (Leica). Dorsal striatal tissue samples were extracted using a pre-chilled metal hollow pipe with an inner diameter of 3 mm. Samples were stored at -80°C before use. The frozen tissues were sonicated in 1% SDS, 1 mM sodium orthovanadate solution (40 Hz, 10 pulses) to prevent undesirable dephosphorylation, and boiled at 95°C for 10 min. Then, 20 μg of total proteins were separated on 4-15% Tris-HCl precast gel (Bio-rad) and transferred onto nitrocellulose membranes (Bio-rad). Membranes were blocked in a 3% bovine serum albumin (BSA) solution for 1h at room temperature, then incubated with primary antibodies (Key Resources Table) in a 3% BSA solution at 4°C overnight. Blots were incubated with secondary anti-rabbit, anti-mouse, anti-chicken or anti-goat IgG DyLight™ 800 or 680 conjugated antibodies (1:5000; Rockland Immunochemicals, Pottstown, PA, USA). The signal from the secondary antibody was acquired using Odyssey Clx (Licor) and quantified using Image Studio Lite (Licor). The signal was normalized to a housekeeping protein used as loading control (β-actin, tubulin or GAPDH depending on the molecular weight of the protein of interest studied) and normalized to the mean value of an internal control sample in each blot.