Mini trans blot transfer cell

Dissected brain regions were homogenised in N-Per Neuronal Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, MA, USA) added with Pierce Protease and Phosphatase Inhibitor Mini Tablets (Thermo Fisher Scientific, Waltham, MA, USA) in glass homogenisers, incubated 10 min on ice and centrifuged at 10,000 g for 10 min at 4 °C. Protein concentration was measured on supernatants with the BCA assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Twenty µg proteins were separated on 4–15% precast polyacrylamide gels (Bio-Rad, Hercules, CA, USA) in Mini-Protean Electrophoresis Cells (Bio-Rad, Hercules, CA, USA), transferred on Immun-Blot PVDF membranes (Bio-Rad, Hercules, CA, USA) in Mini Trans-Blot Transfer Cells (Bio-Rad, Hercules, CA, USA), and blocked in 5% skimmed milk in 0.1% Tween20 Tris-buffered saline (Merck). Incubation with primary antibodies was carried out overnight at 4 °C (Table 2); membranes were washed, incubated with the secondary antibody (Table 2), and revealed and quantified by enhanced chemiluminescence (Clarity ECL Substrate, Bio-Rad, Hercules, CA, USA) in a ChemiDoc instrument (Bio-Rad, Hercules, CA, USA) using the Quantity One 1-D Analysis software (Bio-Rad, Hercules, CA, USA).

Cell lysates were prepared by resuspension of cells in a lysis buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM Na₃VO₄, protease inhibitor cocktail (1 tab/10 ml), and 1% Triton X-100), followed by a 30 min incubation on ice. Lysates were centrifuged at 13,000xg for 30 min at 4°C and the nuclear-free supernatants were used for immunoprecipitation. Immunoprecipitation was performed by pre-adsorption of primary Abs onto protein A-, G-, or protein A/G-coupled beads, based on the differential affinities of the proteins to specific Abs, for 2 h at 4°C. Equal volumes of 2x Laemmli Sample Buffer were added to immunoprecipitates or whole-cell lysates, which were vortexed, boiled for 5 min, and fractionated by SDS-PAGE, either on 10%, or 12.5% polyacrylamide gels, and 16.5% Tris-Tricine gels, using Bio-Rad Mini-PROTEAN II cells. Proteins from the gel were electroblotted onto nitrocellulose membranes (Schleicher and Schuell) at 100V for 1 h, using BioRad Mini Trans-Blot transfer cells. After 1 h of blocking with 3% BSA in TBST at 37°C, the nitrocellulose membranes were incubated in the presence of the indicated primary Abs, followed by incubation with HRP-conjugated secondary Abs. Immunoreactive protein bands were visualized using an ECL reagent and autoradiography.