Anti nicd1

40μg of total protein was used for Western blot analysis. The samples were separated using 10–15% SDS–PAGE. These proteins were then transferred to polyvinylidene difluoride (PVDF, Millipore) membranes. After blocking with 5% non-fat milk, the PVDF membranes were incubated with primary antibodies in blocking buffer overnight at 4 °C. On the following day, PVDF membranes were incubated with the appropriate secondary antibodies for 2 hours at room temperature. After the membranes had been soaked in an enhanced chemiluminescence reagent (Thermo Scientific) for 5 minutes, the blots were visualized using X-ray film. Primary antibodies for PPM1A, anti-NICD1 and p-SMAD 1/5/8 (Cell Signaling Technology) were used in this analyses and β-ACTIN (sigma) antibody was used as a loading control. For the immunoprecipitation assay, 5 μg of either anti-NICD1 or Phospho-SMAD1/5/8 antibody was added to 500μg proteins extracted from control ATDC5 cells or NICD1 plasmid transfected cells in RIPA buffer (25 mM Tris-HCl, pH7.6, 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate). Overnight incubation at 4 °C allowed complexes to form, after which 20 μl of 50% slurry protein A/G-agarose beads (Santa Cruz Biotechnology) was added and incubated at 4 °C for 3 hours. Immunoprecipitates were washed four times in RIPA buffer and analyzed by Western blots.

Antibodies for immunostaining included chicken anti-GFP (1:1000 dilution, Abcam, Cat# ab13970), rat anti-GFP (1:1000, Nacalai Tesque, Cat# GF090R), anti-Sox2 (1:200, Cell Signaling Technology, Cat# 3728), anti-RFP (1:1000, MBL, Cat# PM005), anti-Fabp7 (BLBP, 1:500, Millipore, Cat# ABN14), mouse anti-S100β (1:200, Sigma-Aldrich, Cat# S2657), rabbit anti-S100β (1:500, Abcam, Cat# ab52642), anti-NICD1 (1:200, Cell Signaling Technology, Cat# 4147), anti-GFAP (1:1000, Abcam, Cat# ab4674), anti-Hey1 (1:200, Millipore, Cat# AB5714), anti-Ascl1 (1:200, BD Biosciences, Cat# 556604), anti-PCNA (1:500, Millipore, Cat# NA03), anti-Dcx (1:1000, Abcam, Cat# ab18723), anti-EGFR (1:500, Fitzgerald, Cat# 20-ES04), anti-Vcam1 (1:500, BD Biosciences, Cat# 550547), Hoechst 33342 (1:10000, Molecular Probes).

Extraction of whole cell proteins was conducted using RIPA buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate) containing protease inhibitors (Sigma-Aldrich, SRE0055-1BO). Extraction of cytosolic and nuclear proteins was conducted using the ReadyPrep protein extraction kit (Bio-Rad, 1632089) according to the manufacturer’s manual. Proteins were quantified using Pierce 660 nm Protein Assay Reagent (Thermo Fisher Scientific, 22660). Immunoblot was performed using the protocol as previously described (22 (link)). The lysates were loaded onto 4–20% Mini-PROTEAN^® TGX™ Precast Gels (Bio-Rad, 4561091). The following antibodies were used: anti-β-actin (Santa Cruz Biotechnology, sc-130300), anti-TC1 (Santa Cruz Biotechnology, sc-98165), anti-caspase-3 (Cell Signaling Technology, 9662), anti-cleaved caspase-3 (Cell Signaling Technology, 9664), anti-Notch1 (Cell Signaling Technology, 4380), anti-Notch2 (Santa Cruz Technology, sc-5545), anti-NICD1 (Cell Signaling Technology, 4147), and anti-NICD2 (Sigma-Aldrich, SAB4502022). The dilutions of the antibodies were based on the manufacturers’ instructions. The membranes were developed using SuperSignal West Femto Chemiluminescent Substrate (Thermo Fisher Scientific, 34095). The optical density was analyzed using a BioSpectrum 500 imaging system (Ultra-Violet Products Ltd.).