Ab206996

SMAD3 or SMAD4 were immunoprecipitated following immunoprecipitation kit directions (abcam, ab206996). Cells in suspension were collected by centrifugation and adherent cells were detached with Accutase. Washed cells were resuspended into lysis buffer, put on ice for 1 min and then mixed for 30 mins at 4°C. Cells were centrifuged at 10,000g for 10 mins at 4°C and transferred into a fresh tube. For antibody binding, 5 μL of SMAD3 and SMAD4 antibodies were added to 500 μL of sample in lysis buffer with protease inhibitor cocktail. Sample with antibody was mixed overnight at 4°C. 25 μL/sample of Protein A/G Sepharose^® was washed twice with 1 mL of wash buffer, centrifuging at 2000g for 2 mins. After washes the Protein A/G Beads were suspended as 50% slurry in wash buffer. For bead capture, 25 μL of Protein A/G Sepharose^® bead slurry was added to each tube of antibody/sample mixture for 1 hour at 4°C. Sample was collected by centrifuging at 2000g for 2 mins at 4°C. Samples were washed 3 times with 1 mL of wash buffer, centrifuging between. To elute samples 40 μL of 2x Laemmli buffer was added to the bead/sample mixture, boiled for 5 mins and centrifuged to elute. Samples were saved at −80°C for immunoblotting.

Cells were lysed in RIPA buffer (Abcam, ab206996) and equal amounts of proteins (quantified with Pierce BCA assay kit, Thermo Fisher, 23252) were separated on 4–15% gradient Criterion precast gels (Bio-Rad 567–1084). Proteins were then transferred onto nitrocellulose membranes (Bio-Rad). Western Blots were developed with chemiluminescence HRP substrate (Radiance plus, Azure Biosystems AC2103) on a digital image analyzer, Azure Imager c300. Uncropped western blots are shown in Figure S9.