Chemiluminescence detection kit

The cells were collected with a cell scraper (Corning) at a specified time point post-transfection. After centrifugation for 1 min at 16,000× g, the cell pellets were dissolved in 1 × RIPA buffer. After clarification, the protein concentration was determined using the BCA protein Assay Kit (Beyotime, Shanghai, China), according to the manufacturer’s instructions. The cell lysates were mixed with Laemmli’s sample buffer containing 100 mM dithiothreitol, boiled at 90 °C for 5 min, and centrifuged at 16,000× g for 5 min. Equal amounts of protein samples were subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred to 0.2 µM BIORAD nitrocellulose membrane (BIORAD). After blocking the non-specific antibody binding with 5% skim milk in tris-buffer saline containing 0.1% Tween 20 (20 mM Tris-HCl pH 7.4, 150 mM NaCl), the membrane was incubated overnight with 1 μg/mL primary antibody at 4 °C. After washing, 1:2000 diluted anti-rat or anti-rabbit IgG antibody coupled with horseradish peroxidase (DAKO) was added and incubated at room temperature for 2 h. The proteins were detected using the chemiluminescence detection kit (Amersham Biosciences, Amersham, UK) and medical X-ray film (Fuji film, Tokyo, Japan) according to the manufacturer’s instructions.

SDS-PAGE and Western blot was performed as routine. Cells were lysed in ice cold RIPA buffer containing protease inhibitors, followed by sonication and centrifugation. Supernatant was taken for protein quantification by BCA method, and 10 μg proteins were boiled for 10 min before loaded onto 8% polyacrylamide gel for electrophoresis. After the transfer, blockage, and washes, the PVDF membrane was incubated with anti-E-cadherin antibody (1:1000 dilution), at 4°C, for 12 hrs, and then with a goat anti- mouse or rabbit polyclonal horseradish peroxidase (HRP) conjugated secondary antibody (1:10,000 dilution in 1% milk/TBS) for 2 hrs at room temperature. The blots were established by using a chemi luminescence detection kit (Amersham Biosciences, Piscataway, NJ).

The total protein content of cultured cells was extracted using RIPA buffer containing phenylmethanesulfonylfluoride (PMSF). A BCA protein assay kit (Beyotime, Haimen, China) was used to determine the protein concentration. Proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes. After blocking, the membranes were incubated overnight at 4 °C with diluted (1:300) primary antibodies (polyclonal rabbit anti-polβ; Proteintech). Following extensive washing, membranes were incubated with diluted (1:3000) horseradish peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz). Signals were determined with a chemiluminescence detection kit (Amersham Pharmacia Biotech, Piscataway, NJ). An antibody against β-actin (Santa Cruz Biotechnology) served as an endogenous reference.

The treated cells were lysed on ice in PRO-PREP™ Protein Extraction Solution (iNtRON Biotechnology, Seoul, Korea) for 30 min at 4 °C to prepare the whole-cell lysates. The supernatant fractions were recovered by centrifugation (14,000 × g, 20 min, 4 °C), and the protein concentrations of the lysates were determined using a Bradford protein assay. Samples were prepared with 2-mercaptoethanol and denatured by heating at 95 °C for 3 min. The proteins were separated on 8-12% polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were blocked with 1% bovine serum albumin or 5% skim milk and hybridized with the primary antibody. The protein bands were visualized using a chemiluminescence detection kit (Amersham Pharmacia Biotech, Piscataway, NJ) and a LAS-3000 or LAS-4000 imaging system (FUJIFILM Corporation, Tokyo, Japan) after hybridization with the HRP-conjugated secondary antibody. The band intensities of the western blot data were analyzed using Quantity One software (Bio-Rad Laboratories, Hercules, CA).

6,7,4'-THIF was obtained from Chromadex™ (Irvine, CA, USA), and Dulbecco’s modified eagle medium (DMEM) and MMP-1 antibody were purchased from Thermo Fisher Scientific (San José, CA, USA). Medium 199 (M199) and daidzein were purchased from Sigma-Aldrich (St. Louis, MO, USA), and fetal bovine serum (FBS) was purchased from Gemini Bio-Products (Calabasas, CA, USA). CNBr-Sepharose 4B, [γ-32P]-ATP and the chemiluminescence detection kit were obtained from Amersham Pharmacia Biotech (Piscataway, NJ, USA). The protein assay kit was purchased from Bio-Rad Laboratories (Hercules, CA, USA). MTS solution was from Promega (Madison, WI, USA). Penicillin/streptomycin was purchased from Invitrogen (Grand Island, NY, USA). Primary antibodies recognizing phosphorylated MEK (Ser^217/221), total MEK, phosphorylated SEK1/MKK4 (MKK4, Ser²⁵⁷/Thr²⁶¹), phosphorylated MKK3 (Ser¹⁸⁹)/6 (Ser²⁰⁷), total MKK3, phosphorylated p38 (Tyr^180/182), total p38 and PKCα were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against phosphorylated-ERKs (Tyr²⁰⁴), total ERKs, ERK2, total MKK4, phosphorylated JNK (Thr¹⁸³/Tyr¹⁸⁵), total JNK, JNK2 and phosphorylated PKCα (Ser⁶⁵⁷) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Active PKCα and PKCδ proteins were purchased from Millipore (Bedford, MA, USA).

The lysates prepared from 100 μg of the fat-removed dorsal skin of mice, were centrifuged at 13,000 rpm for 30 min, and aliquots of the supernatant containing 100 μg of proteins were subjected to 10% SDS-PAGE. After electrophoresis, the proteins were blotted onto nitrocellulose membrane. Subsequently, the membranes were blocked by incubation with TBS-T (0.1% Tween 20) containing 5% BSA. The membranes were then incubated with primary antibodies (anti-phospho-ERK, anti-phospho–MEK, anti-MMP-9, or anti-ERK), and washed with TBS-T. Protein bands were visualized using a chemiluminescence detection kit (Amersham Pharmacia Biotech) after hybridization with horseradish peroxidase (HRP)-conjugated secondary antibodies (goat anti-rabbit IgG or goat anti-mouse IgG Ab diluted 1:1000). The relative amounts of proteins were detected using an enhanced chemiluminescence Western Blotting Detection Kit (Amersham, Little Chalfont, Buckinghamshire, UK).