Ecl plus western blotting detection system

MCF-7 cells were obtained from the JCRB Cell Bank (National Institute of Health Sciences, Tokyo, Japan), and cultured in RPMI 1640 (Invitrogen, Carlsbad, CA) medium supplemented with 10% fetal bovine serum (FBS) at 37 °C in 5% CO₂. Western blotting was performed according to the protocol described in Dong et al.^{30 (link)}. Phospho-Erk1/2 (P-Erk1/2) and phospho-Akt (P-Akt) were used as the primary antibodies, and rabbit antibodies against total Erk1/2 (T-Erk1/2), P-Erk1/2, total Akt (T-Akt) or P-Akt (Cell Signaling Technologies, Ipswich, MA) were used as the secondary antibodies after appropriate dilutions (1:200 to 1:1000). The antibody-antigen complexes were detected with horseradish peroxidase-coupled goat antibody against rabbit IgG (Cell Signaling Technologies) after dilution (1:3,000), and then visualized using the ECL-plus Western Blotting Detection System (Amersham Pharmacia Biotech, Arlington Heights, IL).

The cortex was washed thoroughly with ice sold saline 10% (w/v) and later homogenized in a Potter Elvehjem homogenizer in ice-cold 50 mM phosphate buffer at pH 7.4 containing mammalian protease inhibitor cocktail (Halt™ Protease Inhibitor Cocktail, Thermo Fisher Scientific, Waltham, MA, USA). The homogenates were used for the determination of total protein concentration by the Bradford method using bovine serum albumin as the standard. Equal protein concentrations (15 µg) were denatured in gel loading buffer at 85 °C for 5 min and then loaded onto 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes and incubated at 4 °C overnight with the primary antibody diluted in phosphate buffered saline with the detergent Tween^® 20. Housekeeping protein β-actin was used as a loading control. Positive immunoreactive bands were quantified densitometrically and expressed as the ratio of problem to β-actin in arbitrary units. The protein bands were visualized with enhanced chemiluminescence reagents (ECL Plus Western Blotting Detection System, Amersham Pharmacia Biotech, Piscataway, NJ, USA) and analyzed, and their intensity was quantified using a Kodak Electrophoresis Documentation and Analysis System 290 (EDAS 290).

Total proteins were extracted from cells using radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma-Aldrich) and quantified by Bicin Choninic Acid (BCA) protein assay kit (Thermo). The Western blot was performed according to standard procedures. The following antibodies were used: EphA8 (Abcam, USA), TGFβ1, TGFβR1, smad2, smad3 and smad4 (Cell Signaling Technology, Beverly, MA, USA); β-actin (Proteintech, USA) was used as loading control. The blots were incubated with goat anti-rabbit or anti-mouse secondary antibodies (Bioss, Beijing, China). The proteins were visualized using a chemiluminescence method (ECL Plus Western Blotting Detection System; Amersham Biosciences, Foster City, CA, USA). The bands were quantified by ImageJ software.

Melan-a cells (1.5 × 10⁴ cells/ml) were cultured in a 60 mm dish for 24 h. And then, DGD was treated with or without indicated concentration and time. The cell were lysed in cold lysis buffer (20 mM Tris-HCL (PH 7.5), 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM glycerophosphate, 1 μg/ml leupeptin, 1 mM PMSF and a protease inhibitor). The protein concentration was determined using a dye-binding protein assay kit (Bio-Rad Laboratories Inc.), as described in the manufacturer’s manual. A 20-40 μg lysate protein was separated via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to a polyvinylidene fluoride membrane (Millipore Crop., USA). After blotting, the membranes were blocked with 5% non-fat skim milk in a Tris buffered saline-T buffer at 4°C overnight and incubated for 2 h with the specific primary antibodies (1:1000). After hybridization with secondary antibodies (1:5000, Santa Cruz Biotech, USA), the protein bands were visualized using an ECL plus Western blotting detection system (Amersham™, USA).

Cells were lysed in an ice-cold lysis buffer (0.137M NaCl, 2mM EDTA, 10% glycerol, 1% NP-40, 20mM Tris base, pH 8.0) with protease inhibitor cocktail (Sigma). The proteins were separated in 10% SDS-PAGE and further transferred to the PVDF membrane. The membrane was incubated with appropriate antibodies, washed and incubated with HRP-labeled secondary antibodies, and then the blots were visualized using the ECL+plus Western Blotting Detection System (Amersham). The blots were quantitated by IMAGEQUANT, and the results were normalized by β-actin.

Protein extracts were resolved by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (Hybond-P; GE Healthcare, Piscataway, NJ), followed by incubation with the indicated primary and secondary antibodies conjugated to horseradish peroxidase (GE Healthcare). Signals were detected using the ECL detection system (Amersham ECL plus Western Blotting detection system, Fairfield, CT).