Sc 32290

Each gram of hippocampi were homogenized with 30 µl cocktail protease inhibitor (Roche Molecular Biochemicals, Germany) and 3 ml RIPA buffer (US Biological, USA). After centrifugation at 12000 rpm (4 •C), the supernatant was collected and stored at -20 •C. BCA assay and spectrophotometry were used to assess the total protein concentration. The supernatant sample (50µg of protein) was separated by 10% SDS-PAGE and transferred to PVDF membrane (Millipore Inc., Darmstadt, Germany). After blocking with 5% skim milk for 2 hours, the membrane was incubated with primary antibody overnight: Rac1 (1:500, sc-514583, Santa Cruz Biotechnology), Tiam1 (1:500, sc-393315, Santa Cruz Biotechnology), α-chimaerin (1:500, sc-365985, Santa Cruz Biotechnology), Bcr (1:500, sc-104, Santa Cruz Biotechnology), GluR-1 (1:500, sc-13152, Santa Cruz Biotechnology), Synapsin 1(1:500, ab254349, Abcam), and PSD-95 (1:500, sc-32290, Santa Cruz Biotechnology). The speci city of these primary antibodies has been veri ed (Mao et Tsai et al. 2012 (link)). The membrane was incubated with a mixture of anti-rabbit IgG (Golden Bridge, Zhongshan, China) and horseradish peroxidase (HRP) for 1 hour at room temperature. Quantity One software (Bio-Rad, USA) was used for quantitative analysis.