Trixon x

The cells were lysed using lysis buffer (1% phosphatase inhibitor cocktail II, 0.2 mM of PMSF, and 2% Trixon-X in PBS (Sigma-Aldrich, St. Louis, MO, USA)) to obtain the total protein. The lysates were cleared by centrifuging at 13,000 rpm for 5 min at 4 °C. Then, equal amounts of proteins were denatured at 100 °C in an SDS-loading buffer for 10 min. The protein samples (20 µg/well) were subjected to 10% or 8% SDS-PAGE and then were transferred to nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ, USA). The membranes were blocked for 1 h at RT with 5% skim milk in TTBS solution (0.5% TBS-Tween 20), incubated overnight with primary antibodies at 4 °C (Table S4), washed for 6 × 5 min with TTBS, and incubated with a secondary antibody (1:10,000 dilution). Finally, blots were developed using Femto reagent (Thermo Fisher Scientific) and processed using software that measured the densities of protein bands (Fusion Solo, Paris, France).

The total protein from the transfected cells was obtained using a lysis buffer containing 1% phosphatase inhibitor cocktail II, 0.2 mM PMSF, and 2% Trixon-X (Sigma-Aldrich, St. Louis, MO, USA), and protein concentration was determined using a Bradford assay kit (Bio-Rad, Hercules, CA, USA). Protein samples (20 µg) were resolved on SDS-PAGE gels and transferred to nitrocellulose membranes (Amersham, Braunschweig, Germany), which were blocked with 5% skim milk in TBS-Tween 20 (TTBS) for 1 h and then incubated overnight with the indicated primary antibodies (Table S3) at 4 °C, followed by incubation with secondary antibodies (Table S3) for 1 h at room temperature. Blots were developed using a Femto reagent (Thermo Fisher Scientific, Waltham, MA, USA) with a Fusion Solo Chemiluminescence Imaging System (Vilber Lourmat, Marne-la-Vallée, France), and their densities were analyzed with Evolution Capt software (Vilber Lourmat). β-Actin protein levels were used for normalization.

NPCs were dissociated, plated on coverslips pre-coated with poly-ornithine (Sigma, 15 µg/ml in H2O) and fibronectin (Sigma, 2 µg/ml in PBS) and cultured till confluency or certain days into differentiation. Cells were fixed with 4% formalin/PBS solution at room temperature for 10 min and washed with PBS for three times. Fixed cells were permeabilized using 0.4% Trixon-X(Sigma), incubated with primary antibodies at 4 °C overnight, followed by secondary antibodies at room temperature for 60 min. Cells were washed with PBS for three times after both primary and secondary antibodies. Cells were stained with neuronal marker Tuj1 (Covance; 1:1000), oligodendtocyte marker CNPase (Millipore; 1:1000) or astrocyte marker Gfap (Sigma; 1:1000). Hoechst staining was used to label the nuclei. Images were captured using Olympus fluorescence microscope and processed using Imaris and Adobe Photoshop CS6 software.