1protease inhibitor mixture

Cytoplasmic and nuclear protein fractions were isolated from NSCs by means of NE-PER Extraction Kit (Thermo Fisher Scientific). Total extracts from SVZ and hippocampal regions of control mice were lysed in RIPA buffer solution (Pierce, Rockford, IL, USA) containing 50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% sodium deoxycholate, 1% TX-100, 0.1% sodium dodecyl sulfate (SDS) and ×1protease inhibitor mixture (Sigma), 1 mM sodium orthovanadate, and 1 mM NaF. All samples were resolved by Western blot standard procedures^{61 (link)–63 (link)}, using the following antibodies: polyclonal anti-β-Catenin antibody (1:4000, Abcam, Cat. #ab6302), monoclonal anti-actin antibody (1:4000, Sigma, Cat. #T6074), monoclonal anti-GAPDH antibody (1:2000, Abcam, Cat. #ab8245), monoclonal anti-fibrillarin (1:1000, Thermo Scientific, Cat. #MA3-16771). All experiments were repeated independently 3 times.

HCT116 cells and HT-29 cells were lysed in modified RIPA buffer (1% Nonidet P-40, 0.1% sodium deoxycholate, 150 mm NaCl and 1 mm ethylenediaminetetraacetic acid (EDTA) in 50 mm Tris–HCl, pH 7.5) supplemented with 1× protease inhibitor mixture (Sigma-Aldrich). Solubilized proteins were separated by electrophoresis through 10% Tris-glycine gels and transferred onto PVDF membranes. Primary antibodies used for western analysis were rabbit polyclonal anti-SOX9 (Millipore), mouse monoclonal anti-NF-YA (Santa Cruz Biotechnology) and mouse monoclonal anti-β-actin (Santa Cruz Biotechnology). Secondary antibodies used were horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit (Santa Cruz Biotechnology). For co-immunoprecipitation, cells were lysed in RIPA buffer and immunoprecipitated overnight at 4°C with 2 μg antibody and 20 μl protein-A/G agarose beads (Santa Cruz Biotechnology). Bead-bound complexes were washed, eluted and visualized by western blotting.