Ab32397

Cell lysates were lysed by RIPA buffer (Sigma-Aldrich, USA) and moved into tubes for later use. After being washed with PBS twice, the proteins from cells were extracted. Proteins were partitioned on a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Next, the cells were moved to a PVDF membrane (Millipore, Billerica, MA, USA). Blots were carried out with anti-Caspase-3, dilution, 1:500, Abcam, ab13585; anti-Caspase-8, dilution, 1:1,000, Abcam, ab32397; anti-Caspase-9, dilution, 1:2,000, Abcam, ab202068; anti-Ki67, dilution, 1:5,000, Abcam, ab92742; anti-Cyclin D1, dilution, 1:10,000, Abcam, ab134175; anti-DLL1, dilution, 1:500, Abcam, ab10554; anti-DLL3, dilution, 1:500, Abcam, ab63707; anti-Notch1, dilution, 1:500, Abcam, ab65297; anti-HES1, dilution, 1:500, Abcam, ab108937; anti-GAPDH, dilution, 1:10,000, Abcam, ab181602. After that, horseradish peroxidase-conjugated secondary antibodies (bs-0293M; BIOSS, Beijing, China) were added to the cells and incubated at room temperature for 1 h. The bolts were analyzed by chemiluminescent reagents (Millipore, Billerica, MA, USA).

Proteins were extracted by radioimmunoprecipitation assay (Biyuntian Biotechnology Co., Shanghai, China) with 1% phenylmethylsulfonyl fluoride and 1% phosphate inhibitor. Proteins were fractionated in 3-morpholinopropane-1-sulfonic acid/sodium dodecyl sulfate running buffer (Thermo Fisher Scientific) with a protein electrophoresis meter (Bio-Rad) and transferred to poly(vinylidene) fluoride membranes (Bio-Rad) with a Trans-Blot (Bio-Rad). The antibodies used were GAPDH (ab9485, Abcam, Cambridge, UK), caspase-8 (ab32397, Abcam), poly(adenosine diphosphate-ribose) polymerase (PARP) (ab32064, Abcam), BAX (ab32503, Abcam), cyclin E1 (ab33911, Abcam), cyclin D1 (ab134175, Abcam), cyclin B1 (ab32053, Abcam), CDK4 (ab108357, Abcam), CDK6 (ab124821, Abcam), p21 (ab109520, Abcam), N-cadherin (ab18203, Abcam), E-cadherin (ab15148, Abcam), PI3 kinase p110α (4255S, Cell Signaling Technology, Danvers, MA), AKT (9272S, CST), phospho-AKT (Ser473) (4060S, Cell Signaling Technology), c-Myc (9402S, Cell Signaling Technology), phospho-c-Myc (Ser62) (13748S, Cell Signaling Technology), and PRDX2 (10545-2-AP, Proteintech, Rocky Hill, NJ). We used GAPDH as an internal reference.