Anti notch1 icd

Cells were lysed in cell lysate, and then centrifuged at 12,000 × g for 20 min at 4 °C. The supernatant was collected and denatured. Proteins were separated in 10% SDS-PAGE and blotted onto polyvinylidene difluoride membrane (PVDF). The PVDF membrane was treated with TBST containing 50 g/L skimmed milk at room temperature for 4 h, followed by incubation with the primary antibodies: anti-CTR2 (1:200, Novusbio), anti-BCL-2 (1:500, Immunoway), anti-OCT-4 (1:1000, Proteintech), anti-KLF4 (1:500, Proteintech), anti-MDR1 (1:200, Santa),, anti-Notch1 ICD (1:1000, Abcam), anti-Notch2 ICD (1:1000, Abcam), anti-Jagged1 (1:500, Abcam), anti-Jagged2 (1:1000, Abcam), anti-DLL1 (1:500, Abcam), anti-DLL4 (1:500, Abcam), anti-Notch ICD (1:1000, Cell Signaling), anti-SDF-1 (1:1000, Abcam), anti-CXCR4 (1:2000, Abcam) and anti-β-actin (1:1000, Cell signaling) respectively, at 37 °C for 1 h. Membranes were rinsed and incubated for 1 h with the correspondent peroxidase-conjugated secondary antibodies. Chemiluminent detection was performed with the ECL kit (Pierce Chemical, Rockford, IL, USA). The amount of the protein of interest, expressed as arbitrary densitometric units, was normalized to the densitometric units of ß-actin.