Co-immunoprecipitation

Methods are as previously reported [13]. Cells were washed twice in ice-cold PBS, and then transferred into 1.5 mL eppendorf tubes chilled on ice. Cell pellets were then subjected to cytoplasmic and nuclear fractionation using the NE-PER (Pierce). Protein concentration was determined using the Bio-Rad Protein Assay. 200μg of nuclear protein from each treated sample were diluted with 1mL of co-immunoprecipitation buffer (25mM Tris, HCl, pH7.5, 150mM NaCl, 5%glycerol, 0.5%NP40, 1mM EDTA, 2mM DTT, 1X protease inhibitor cocktail). 2μg of CBP, p300, or normal rabbit IgG antibody were added to the diluted protein sample and incubated at 4°C on a rotating mixer overnight. 50μl of a 50% slurry of protein A-agarose beads, equilibrated in co-ip buffer, were added to each diluted sample containing antibody. The protein-antibody complexes were then allowed to bind to protein A-agarose on a rotating mixer for 1 h at 4°C. Protein A-agarose beads containing bound antibody/protein complex were washed three times using 500μl of co-ip buffer each time. After the final wash, the agarose beads were resuspended in 30μl of 2X Laemmli Buffer and boiled for 10 minutes for SDS-PAGE. For proteomic analysis, the agarose beads were suspended in 5% Sodium Deoxycholate (SDC) (Sigma-Aldrich). The supernatant, containing bound protein complexes were separated from the agarose beads using Illustra microspin columns. The boiled supernatants were loaded onto 8% polyacrylamide gels and subjected to SDS-PAGE. Electrophoresed proteins were then transferred onto PVDF membrane overnight under low voltage (25V) in a cold room. PVDF membranes were then probed using a mouse monoclonal antibody for β-catenin (1:5000 dilution). Detection of β-catenin was performed on the Bio-Rad ChemiDoc MP Imaging System.