Tiam1

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.

The NPC cells were harvested and washed twice with phosphatebuffered saline (PBS). The protein concentration of cell lysate was determined using the Pierce BCA protein assay kit (Thermo Fisher Scienti c, China). 30 mg of proteins were loaded on premade 8-15% SDS polyacrylamide gel for separation and then electrotransferred onto a nitrocellulose membrane. The blots were incubated in PBS buffer with 5% defat milk and 0.02% Teween-20 at room temperature for 1 h before primary antibody incubation overnight at 4C. The antibodies including β-catenin, β-actin, MMP-7, Fibulin-3, Tiam1, E-cadherin, Snail, Slug, Vimentin were obtained from Santa Cruz Biotechnology Inc., US and applied with 1:1000 dilution. The SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scienti c, China) was used for visualization of immunoreactive proteins.