Sc 481

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS),
phosphate buffer saline (PBS), penicillin and streptomycin were
obtained from Hyclone (Logan, UT, USA).
Lipofectamine^®RNAiMAX and TRIzol® Reagent was purchased
from Invitrogen (Carlsbad, CA, USA). Ribonuclease A (RNase A) and
DNAse were purchased from Sigma-Aldrich (St. Louis, MO, USA). The
antibodies used were the following: rabbit anti-Inhibin beta B
polyclonal antibody (17577-1-AP, 1:200) purchased from Proteintech
Group (Chicago, IL, USA); anti-bax (BS-2538, 1:800) purchased from
Bioworld Technology (St. Louis Park, MN, USA); anti-cyclin D1 (sc-753,
1:200), anti-cyclin E (sc-481, 1:200), anti-Bcl-2 (sc-783, 1:200 and
anti-GAPDH (sc-59540, 1:3,000) ) purchased from the Santa Cruz
Biotechnology (Dallas, TX, USA).

HCT116 cells (2×10⁶ cells/well) were cultured overnight in a 6-well plate and treated with cardamonin (0, 20, 40 and 80 µM) for 72 h. Cell lysates were prepared by the addition of 100 µl lysis buffer for 15 min. Protein content was determined using the BCA method. A total of 50 µg protein per lane was separated by 8–10% SDS-PAGE and electrotransferred onto polyvinylidene difluoride membranes. The membranes were blocked with TBST containing 5% non-fat dried milk at 37°C for 1 h and incubated with specific primary antibodies targeted at B-cell lymphoma-associated X (Bax, sc-6236, 1:1,000), Myc proto-oncogene protein (c-MYC, sc-789, 1:1,000), octamer-binding transcription factor 4 (Oct4, sc-9081, 1:1,000), and cyclin E (sc-481, 1:1,000) (all from Santa Cruz Biotechnology, Inc.), testes-specific protease 50 (TSP50, ab181993, 1:1,000; Abcam), NF-κB (sc-101749, 1:1,000) and GAPDH (sc-25778, 1:1,000) (both from Santa Cruz Biotechnology, Inc.) overnight at 4°C. Following three washes with TBST, membranes were incubated with anti-rabbit or mouse horseradish peroxidase-conjugated secondary antibodies (sc-2004 or sc-2005, 1:5,000; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. Signals were detected by enhanced chemiluminescence (ECL Plus detection system) and band density was analyzed using Carestream MI software (Carestream Health, Inc., Rochester, NY, USA).