Invitrogen polyvinylidene difluoride

Whole cell extracts from cultured cells were prepared using radioimmunoprecipitation assay buffer (Thermo Scientific, Rockford, IL) containing protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland). Protein quantification was done using a BCA protein assay kit (Thermo Scientific) according to the manufacturer's instructions. Total protein (20 μg) was loaded onto 4–12% bis–tris gels with 3-(N-morpholino) propanesulfonic acid buffer and separated by a NuPAGE electrophoresis system (Invitrogen). Protein was transferred to Invitrogen™ polyvinylidene difluoride and immunoblotting was carried out according to standard protocols. Monoclonal antibody against PMS2 was purchased from Origene Technologies (Rockville, MD) and monoclonal antibody against GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA) was used to confirm equal loading. The membrane was washed and then incubated with secondary antibodies conjugated to horseradish peroxidase (Cell Signaling Technology). Protein complexes were visualized with the Echo-chemiluminescence (ECL) Detection System (GE Healthcare, Little Chalfont, UK) using the Chemidoc Imaging System (Bio-Rad Laboratories, Hercules, CA).

Whole cell extracts from cultured cells were prepared using radioimmunoprecipitation assay buffer (Thermo Scientific, Rockford, IL) containing protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland). Protein quantification was done using a BCA protein assay kit (Thermo Scientific) according to the manufacturer's instructions. Total protein (20 μg) was loaded onto 4-12% bis–tris gels with 3-(N-morpholino) propanesulfonic acid buffer and separated by a NuPAGE electrophoresis system (Invitrogen). Protein was transferred to Invitrogen™ polyvinylidene difluoride and immunoblotting was carried out according to standard protocols. Monoclonal antibodies against MLH1, cleaved PARP, c-Abl, and phosphorylated c-Abl were purchased from Cell Signaling (Danvers, MA) and monoclonal antibody against GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA) was used to confirm equal loading. The membrane was washed and then incubated with secondary antibodies conjugated to horseradish peroxidase (Cell Signaling Technology). Protein complexes were visualized with the Echo-chemiluminescence (ECL) Detection System (GE Healthcare, Little Chalfont, UK) using the Chemidoc Imaging System (Bio-Rad Laboratories, Hercules, CA).