Protein Expression Analysis of Notch Signaling Pathway

Total protein was extracted using RIPA buffer (KeyGen, China). Protein concentration was quantified using a BCA protein kit (Beyotime, China). Proteins (30 µg) were separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After blocking with 5% milk, membranes were incubated with primary antibodies overnight at 4°C, including Numb (ab155415, Abcam, USA), Numbl (ab37500, Abcam, USA), Notch1 (ab128076, Abcam, USA), Hes1 (ab71559, Abcam, USA), and β-actin (ab8227, Abcam, USA). Subsequently, the samples were incubated with secondary antibodies (Abcam, USA) for 1.5 h at room temperature and analyzed using enhanced chemiluminescence reagent (Beyotime, China) [28 (link)].