Western breeze chromogenic immunodetection system

Western blot analysis was performed according to standard procedures. Equal amounts of protein (40 μg) from each sample were separated on SDS-PAGE precast gels (Invitrogen Life Technologies) and transferred onto nitrocellulose membranes (Invitrogen Life Technologies). After blocking with 5 % skim milk, the membranes were blotted with anti-bodies against β-catenin, c-Myc, Bax, Bcl-2, GADPH (all from Cell Signaling Technology, USA) or Dvl-1(Santa Cruz, USA) by using the Western Breeze chromogenic immunodetection system (Invitrogen Life Technologies),the images were captured and quantified with Image Lab 4.0 software (Bio-Rad Laboratories, USA).

After treatment, H9C2 cells were lysed in cold RIPA buffer (Thermo, USA), added with Halt Protease Inhibitor Cocktail (Thermo, USA). After centrifugation at 14,000 g for 15 min at 4 °C, the supernatants were collected, and the total protein content was measured spectrophotometrically using the BCA Protein Assay kit (Thermo, USA). Western blot analysis was performed according to standard procedures. Equal amounts of protein (40 μg) from each sample were separated on SDS-PAGE precast gels (Invitrogen, USA) and transferred to nitrocellulose membranes (Invitrogen, USA). After blocking with 5 % skim milk, the membranes were blotted with antibodies against β-catenin, c-Myc, Bax, Bcl-2, GADPH (all from Cell Signaling, USA) or Dvl (Santa, USA) using the Western Breeze chromogenic immunodetection system (Invitrogen, USA). The images were captured and quantified with Image Lab 4.0 software (Bio-Rad Laboratories), and the values were normalized to GAPDH.