Ecl western blotting substrate

Nitrocellulose membrane (0.2 µm, Bio-Rad) was soaked in 0.01 M phosphate buffered saline (PBS) for 30 min before use. A solution of protein (1 µM, final concentration) was mixed with the drug solution (2 µM in 5% DMSO/PBS, final concentration) or vehicle. After 1 h incubation at room temperature, 90 µL of the mixture was immobilized on the membrane using a 96-well Bio-Dot Microfiltration Apparatus under vacuum. Each strip including 5–6 duplications was assigned to the corresponding antibody recognition for the epitope of interest (EOI). Briefly, the membrane was blocked with 5% nonfat milk for 1 h at room temperature and incubated with a primary antibody (1 : 2000 diluted with 5% nonfat milk) at 4 °C overnight. After washing with 0.1% Tween 20 in TBS (TBS-T) for 3 × 10 min, the membrane was incubated with HRP-conjugated secondary antibody (1 : 2000 diluted with 5% nonfat milk) for 1.5 h at room temperature and washed with TBS-T for 3 × 10 min. Visualization of EOI was performed with an enhanced chemiluminescent reagent (Pierce ECL Western Blotting Substrate) using an IVIS®Spectrum imaging system (PerkinElmer, Hopkinton, MA) with a blocked excitation filter and an opened emission filter. The chemiluminescence readout from the control group (without the tested drug) was normalized to 1.0, and the alteration effect for the EOI was quantified using the ratio between the two groups.

Brains of mice were homogenized with lysis buffer (1 mM HEPES, 5 mM benzamidine, 2 mM 2-mercaptoethanol, 3 mM EDTA, 0.5 mM MgSO₄, 0.05% NaN₃, protease inhibitor cocktail III, phosphatase inhibitor cocktail II), and centrifuged at 100,000×g for 1 h to obtain cytosolic and particulate (membrane bound) fractions. The fractions were loaded onto 4–12% Bis-Tris SDS-PAGE gels (Invitrogen), transferred onto PVDF membranes, and blocked with bovine serum albumin. After an incubation with indicated primary antibodies, the membranes were incubated with HRP-conjugated secondary antibodies (American Qualex, San Clemente, CA), and reacted with ECL western blotting substrate (PerkinElmer, Waltham, MA). Chemiluminescence detection and densitometry analysis were performed using Versadoc XL imaging apparatus and Quantity One (Bio-rad, Hercules, CA).