Re blot plus western blot recycling kit

Cells were lysed at 4 °C with RIPA Lysis Buffer (Upstate, Lake Placid, NY). Equal amounts of proteins (20 μg per lane) were separated by NuPAGE 4–12 % Bis-Tris Gel (Invitrogen, Carlsbad, CA) electrophoresis. Protein fractions were then electrophoretically transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with Blocker/Diluent solution (Western Blot Kit, Invitrogen). Then the membrane was incubated with rabbit polyclonal antibody to human beta-catenin (Cell Signaling Technology, Inc., Danvers, MA) for 1 hour at room temperature. After washing in wash buffer (Western Blot Kit, Invitrogen), the membrane was incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin G (Western Blot Kit, Invitrogen) for 1 hour at room temperature. The antigen antibody-peroxidase complex was visualized using the ECL chemiluminescence solution (Western Blot Kit, Invitrogen). The blot was subsequently stripped with Re-Blot Plus Western Blot Recycling Kit (Chemicon International, Inc., Temecula, CA) and rehybridized with an anti-GAPDH antibody (Santa Cruz Biotechnology, INC.) as a control for protein loading. Densitometric quantitation was performed using the ImageJ software (NIH, Bethesda, MD). Band intensity values of beta-catenin were standardized to those of GAPDH.