Sc 65480

Cells were washed with ice-cold PBS and lysed in RIPA buffer [50 mmol/l Tris (pH 7.5), 150 mmol/l NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS)] containing phenylmethylsulfonyl fluoride (PMSF; 1 mmol/l) and protease inhibitors (2 g/ml; Protease inhibitor cocktail set III, Calbiochem, Billerica, MA, USA) on ice for 30 min. The lysates were clarified by centrifugation at 13,000 × g for 30 min at 4°C. The total protein concentration was estimated using a Protein Assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel, transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA), blocked and probed with antibodies against β-catenin (1:1,000; sc-65480; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), c-myc (1:1,000; sc-40; Santa Cruz Biotechnology, Inc.), Jagged (1:1,000; sc-390177; Santa Cruz Biotechnology, Inc.), Notch1 (1:1,000; sc-373891; Santa Cruz Biotechnology, Inc.), Notch2 (1:1,000; sc-5545; Santa Cruz Biotechnology, Inc.), DLL4 (1:1,000; ab7280; Abcam, Cambridge, MA, USA) and Pra-1 (1:1,000; ab76413; Abcam). Upon washing, blots were incubated with horseradish peroxidase-conjugated secondary antibodies and visualized by super enhanced chemiluminescence detection reagent (Applygen, Beijing, China).

Endometrial stromal cells were lysed with lysis buffer (radioimmunoprecipitation assay buffer) containing protease inhibitor cocktail (phenylmethanesulfonyl fluoride) and EDTA at 4°C for 30 min. Cells were centrifuged at 12,000 × g for 10 min at 4°C. Subsequently, the concentration of total protein was determined using the BCA assay, and 40 µg/lane total protein was subjected to 10–12% SDS-PAGE and subsequently transferred onto a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Inc.). The membrane was blocked with 5% non-fat milk in TBS containing 0.1% Tween-20 for 1 h at 37°C, and subsequently incubated with antibodies against Wnt (sc-376029; 1:1,000), β-catenin (sc-65480; 1:1,000), ZEB1 (sc-515797; 1:1,000), apoptosis regulator BAX (Bax; sc-20067; 1:1;000) and GAPDH (sc-51631; 1:5,000; all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4°C. Following this, membranes were incubated with anti-rabbit immunoglobulin G secondary antibody (sc-2004; 1:5,000; Santa Cruz Biotechnology, Inc.) at 37°C for 1 h and were developed using an enhanced chemiluminescence kit (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). Protein levels were quantified using Bio-Rad Laboratories, Inc. Quantity One software (version 3.0).

Cells were cultured in 6-well plates at 2×10⁶/well and transfected with Wnt10a-siRNA fragments or treated with 0.4 nM LGK-974, a specific inhibitor for porcupine homolog (Drosophila; PORCN), in Wnt/β-catenin signaling pathway. After 48 h of incubation at 37°C, total protein was extracted using iced lysis buffer (1% Triton X-100, 50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 0.1% SDS; 1 mM phenylmethanesulfonyl fluoride; 1 mM EDTA). Subsequently, the total protein concentration was determined using a bicinchoninic acid assay (BCA Protein Assay kit; Generay Biotech Co., Ltd., Shanghai, China). A total of 10 µg protein was separated on an 10% SDS-PAGE gel, transferred to nitrocellulose membranes followed by blocking with 5% non-fat milk for 2 h at room temperature, and then incubated overnight with primary mouse monoclonal antibodies against β-catenin (1:250, sc65480; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), cyclin D1 (1:400, sc-70899; Santa Cruz Biotechnology, Inc.), LEF1 (1:800, ab137872), TCF4 (1:500, ab217668) and GAPDH (1:2,000, ab181602) (Abcam, Cambridge, MA, USA) at 4°C. Membranes were subsequently washed and incubated with a horseradish peroxidase-conjugated secondary antibody (1:10,000, ab97051) for 1 h at 25°C. Immunoreactivity was determined using an enhanced chemiluminescence kit (Merck KGaA).