Kaleidoscope molecular weight markers

Cells or brain tissues were homogenized with an ultrasonicator (GE 130PB, Cole-Parmer, Vermon Hills, IL, USA) in 0.05 M HCl-Tris, pH 9.0 buffer, in the presence of a protease inhibitor cocktail (Mini-complete, Roche, Mannheim, Germany). The extracts were centrifuged (12,500 rpm, 15 min) and the supernatants collected. Total proteins were determined by the Bradford assay (Bio-Rad, Hercules, CA, USA), and 50 µg of protein/lane was separated in 12.5% SDS-PAGE slabs, and then transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA) as previously described [53 (link)]. For immunoblotting, membranes were blocked with 5% non-fat milk (Bio-Rad) in TBS for 2 h. Then, membranes were incubated overnight at RT with the corresponding primary antibodies (Table 1) in TTBS. After washing the membranes with TTBS (3 × 10 min), they were incubated for 2 h with the corresponding HRP-conjugated secondary antibodies. Immunoreactive bands were visualized using ECL blotting detection reagent (Amersham-Pharmacia, Buckinghamshire, UK) on autoradiography film (Fujifilm, Tokyo, Japan). Kaleidoscope molecular weight markers (Bio-Rad) were used as reference for molecular mass determination.

Hippocampal cell cultures (4 wells per experimental group) were homogenized with an ultrasonicator (GE 130PB, Cole-Parmer, Vermon Hills, IL, USA) in 0.05 M HCl-Tris, pH 9.0 buffer, in the presence of a protease inhibitor cocktail (Mini-complete, Roche, Mannheim, Germany). The extracts were centrifuged (12,500 rpm, 15 min), and the supernatants were collected. Total proteins were determined by the Bradford assay (Bio-Rad, Hercules, CA, USA), and 50 μg of protein/lane was separated in 12.5% SDS-PAGE slabs and then transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA) as previously described [24 (link)]. For immunoblotting, membranes were blocked with 5% nonfat milk (Bio-Rad) in TBS for 2 h. Then, membranes were incubated overnight at RT with the corresponding primary antibodies (Table 1) in TTBS. After washing the membranes with TTBS (3 × 10 min), they were incubated for 2 h with the corresponding HRP-conjugated secondary antibodies. Immunoreactive bands were visualized using the ECL blotting detection reagent (Amersham-Pharmacia, Buckinghamshire, UK) on autoradiography film (Fujifilm, Tokyo, Japan). Kaleidoscope molecular weight markers (Bio-Rad) were used as reference for molecular mass determination.