Immobilon polyvinyl difluoride pvdf membrane

Cells were washed with PBS, harvested, and lysed in 0.2% NP-40 lysis buffer. The protein lysates were subjected to immunoprecipitation with antibodies containing anti-Smad4 or anti-β-catenin (Abcam, MA) overnight, and then incubated with protein A/G Plus Agarose (Fisher) for 4 hours. The immunocomplex was washed with PBS three times, followed by mixture with SDS buffer. For western blotting, whole cell or co-precipitated extracts were separated by a Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), blotted onto Immobilon polyvinyl difluoride (PVDF) membrane (Millipore, MA), and incubated with primary antibodies containing anti-Trb3, anti-Smad4, anti-β-catenin or anti-GAPDH. The membranes were subsequently incubated in secondary antibodies conjugated with Horseradish Peroxidase (HRP) (Millipore, MA) and visualized with chemiluminescent HRP (Denville Scientific, NJ).