Quantity one software version 3.0

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).

At 0, 2, 8, 12 and 24 h post-treatment, the burn tissue samples were collected and homogenized, and centrifuged at 8,000 × g for 15 min at 4°C. The supernatant was used to measure total protein concentrations using a BCA kit. The proteins (50 µg) were separated using 10% SDS polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. The membrane was blocked with 5% milk in TBST for 1 h at 4°C and incubated with the following antibodies: NF-κB/p65 (cat. no. sc-109, 1:2,000; Santa Cruz Biotechnology, Inc., Franklin Lakes, NJ, USA), vascular endothelial growth factor (VEGF; cat. no. sc-13083, 1:4,000; Santa Cruz Biotechnology, Inc.), protease-activated receptor 1 (PAR1; cat. no. SAB5300042, 1:3,000; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and β-actin (cat. no. sc-7210, 1:5,000; Santa Cruz Biotechnology, Inc.) at 4°C overnight. Subsequently, the membrane was incubated with sheep anti-rabbit or mouse IgG (cat nos. 14708 or 14709, 1:1,000; Cell Signaling Technology, Inc.) at room temperature for 2 h, and bands were revealed using an ECL kit (Cell Signaling Technology, Inc., Danvers, MA, USA) and quantified using Quantity One software version 3.0 (Bio-Rad, Hercules, CA, USA).