Fusion fx

Primary cortical neurons were lysed using Radio-immunoprecipitation assay buffer (RIPA) buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 1 mM Na3VO4, 5 mM NaF, and a protease inhibitor cocktail). Lysates were centrifuged at 15,000× g for 20 min at 4 °C, and the supernatant was transferred to fresh tubes. The obtained protein was measured by a BCA assay kit (Bio-Rad, Hercules, CA, USA).
Equal amounts of protein were heated in a 4X protein-loading buffer, and were subjected to SDS-PAGE for protein size separation. After gel electrophoresis, proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, MA, USA) and blocked in 5% skimmed milk in 1X TBST for 1 h. The membranes were probed in primary antibody for overnight at 4 °C and washed three times with 1X TBST (1X TBS, 0.1% Tween-20), and then incubated in appropriate secondary antibodies. After washing three times in 1X TBST, the protein bands were detected using Western HRP substrate (Luminata Forte, Millipore) in a chemiluminescence imaging system (Fusion FX, Vilber Lourmat, Collégien, France). The antibodies used are listed in Table 1.

Treated whole cells were harvested and lysed in RIPA lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) supplemented with 1 mM NaF, 1 mM Na₃VO₄ and a protease inhibitor cocktail (Sigma–Aldrich, USA). Total protein content was quantified using a BCA protein assay kit (Thermo Fisher Scientific, USA) following the manufacturer’s instructions. Total protein at 25∼30 μg for each sample was separated by SDS–PAGE; the proteins were then transferred onto PVDF or nitrocellulose membranes. The membranes were blocked in 5% skimmed milk for 1 h and at room temperature (RT) and were then incubated with primary antibodies overnight at 4°C. Anti-Jagged1 and anti-phospho-Stat5a/b (Tyr694/699) antibodies were purchased from Santa Cruz Biotechnology. Anti-cleaved Notch1 (Val1744), anti-phospho-Stat3 (Tyr705), anti-cleaved PARP (Asp214), anti-cleaved caspase 3 (Asp175) and anti-GAPDH antibodies were obtained from Cell Signalling Technology. Following the incubation with the primary antibodies, the membranes were washed and probed with HRP-conjugated secondary antibodies 1 h at RT. The bound antibodies were detected with a chemiluminescent reagent (Millipore, USA) using an enhanced chemiluminescence imaging system (Fusion FX, Vilber Lourmat, France).