Rabbit anti notch1

The primary antibodies used were mouse anti-GFP (Invitrogen—1:1000), rabbit anti-GFP (Abcam—1:5000), chicken anti-GFP (Millipore—1:1000), mouse anti-SatB2 (Abcam—1:400), mouse anti-βIII-tubulin (Covance—1:1000), rabbit anti-βIII-tubulin (Covance—1:1000), rabbit anti-Pax6 (Covance—1:2000), mouse anti-Pax6 (Abcam—1:250), rabbit anti-GAPDH (Sigma—1:1000), rabbit anti-Tbr2 (Abcam—1:500), rabbit anti-Erk (Santa Cruz Biotechnology—1:1000), rabbit anti-Notch1 (Abcam—1:250), rabbit anti-RFP (MBL—1:1000), mouse anti-Actin (Sigma—1:2000), mouse anti-MG-H1 (Cell Biolabs—1:50), mouse anti-GAPDH (Abcam—1:1000), rabbit anti-GLO1 (Abcam 1:500), mouse anti-puromycin (Kerafast—1:1000). The donkey anti-mouse and anti-rabbit Alexa 488, 555 and 647-conjugated secondary antibodies were obtained from ThermoFisher and used at 1:500 dilutions. HRP-conjugated goat anti-mouse, anti-rabbit or anti-chicken secondary antibodies were purchased from ThermoFisher and used at 1:5000 dilutions. NIR-conjugated goat anti-mouse and anti-rabbit secondary antibodies were acquired from LI-COR and used at 1:25,000 dilutions.

The NSCs were lysed in RIPA (CWBIO, Beijing, China) lysis buffer, and the total proteins were fractionated by 8–15% SDS-PAGE (polyacrylamide gel electrophoresis). The membranes were blocked with 5% BSA. Before incubation with secondary antibody, membranes were incubated with the following primary antibodies: rabbit anti-RNF220 (Sigma-Aldrich, Cat# HPA027578), rabbit anti-ZC4H2 (Sigma-Aldrich, Cat# HPA049584), mouse anti-Sin3B (Santa Cruz, Cat# sc-13145), rabbit anti-Cend1 (Abcam, Cat# ab113076), rabbit anti-Neurogenin1 (Abcam, Cat# ab66498), mouse anti-p53 (Abcam, Cat# ab26), rabbit anti-Hes1 (Abcam, Cat# ab108937), rabbit anti-Notch1 (Abcam, Cat# ab52627), mouse anti-Six3 (Rockland, Cat# 35944), mouse anti-Neuritin (Santa Cruz, Cat# sc-365538), rabbit anti-Ascl1 (BD Pharmingen, Cat# 556604), rabbit anti-CyclinD1 (Cell Signaling Technology, Danvers, MA, USA, Cat# 2922S), mouse anti-CyclinB1 (Cell Signaling Technology, Cat# 4135S), rabbit anti-P21 (Abcam, Cat# ab109199), and mouse anti-α-tubulin (ProteinTech, Rosemont, IL, USA, Cat# 11224-1-AP). Band intensities were quantified using ImageJ software and normalized to α-tubulin.

Cells were washed twice with phosphate‐buffered saline and lysed in RIPA buffer containing 1 mM phenylmethylsulfonyl fluoride (Beyotime Institute of Biotechnology, Shanghai, China, http://www.beyotime.com) on ice [33]. The cell lysates were centrifuged at 12,000g for 15 minutes at 4°C, and the supernatants were collected. Equal amounts of protein (20 μg) were fractionated using SDS‐polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (EMD Millipore, Billerica, MA, http://www.emdmillipore.com). The membranes were blocked in 5% bovine serum albumin in Tris‐buffered saline with Tween‐20 at room temperature for 1 hour and probed with primary antibodies at 4°C overnight. The next day, membranes were incubated with secondary antibodies for 1 hour at room temperature and visualized using enhanced chemiluminescence (EMD Millipore). The following antibodies were used: rabbit anti‐Notch 1 (Abcam, Cambridge, MA, http://www.abcam.com), rabbit anti‐β‐catenin (Cell Signaling Technologies, Boston, MA, http://www.cellsignal.com), rabbit anti‐Gli1 (Abcam), rabbit anti‐cyclin E1 (Abcam), mouse anti‐β‐actin (Santa Cruz Biotechnology, Dallas, TX, http://www.scbt.com), and goat anti‐mouse and mouse anti‐rabbit (Cell Signaling Technologies).