Anti notch 1 sc 373891

Total protein was extracted from cells using RIPA kit (Pierce Biotechnology, Rockford, IL, USA) followed by the BCA protein assay to determine protein concentration. Equal amounts of protein were electrophoresed on a polyacrylamide gel and transferred onto polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). The membrane was incubated with anti-leptin, anti-Notch-1 (sc-373891), anti-total (t)-JAK1, anti-t-JAK2, JAK1 phospho (p)-Tyr1022/1023, JAK2 p-Tyr1007/1008, anti-t-STAT3, STAT3 p-Tyr705, and anti-β-actin, all of which were purchased from Santa Cruz Biotechnology. After washes, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling, Beverly, MA, USA). Bound antibody was detected with enhanced chemiluminescence (ECL) kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). β-actin served as an internal control.

The target gene fragments were amplified by PCR and were inserted into pcDNA3.0 (Invitrogen) to construct eukaryotic expression vectors. The target sequence of BEND5 shRNA was 5'-GCAAATACGTCGTCCTATT-3'. The human breast cancer cell lines MDA-MB-231 and ZR75-1, and human embryonic kidney cell line HEK293T were purchased from the American Type Culture Collection. Anti-BEND5 (20931-1-AP), anti-HEY2 (10597-1-AP), anti-E-cadherin (20874-1-AP), anti-N-cadherin (22018-1-AP), anti-Vimentin (10366-1-AP), and anti-MAML1 (55493-1-AP) were purchased from Proteintech. Anti-HES1 (ab71559) was purchased from Abcam. Anti-BEND5 (NBP2-26211) was from Novus Biologicals. Anti-RBPJ (sc-271128), anti-Notch1 (sc-373891), and anti-β-actin (sc-47778HRP) were purchased from Santa Cruz Biotechnology. Anti-FLAG (A8592), anti-FLAG M2 agarose (A2220), anti-c-MYC gel (E6654), and anti-c-MYC-peroxidase (A5598) were from Sigma-Aldrich. Dulbecco's modified Eagle's medium (DMEM, 12491-015) was purchased from Gibco. VigoFect reagent (T001) was purchased from Vigorous Biotechnology. CCK8 reagent (CK04) was purchased from Dojindo. Paraformaldehyde (BL539A) was purchased from Biosharp.