Ep769y

Cells pellet was lysed in RIPA buffer (Boston Bioproducts) and subjected to SDS-polyacrylamide tris-glycine gel separation (Invitrogen). And the membrane transfer was performed using iBlot® 7 Minute Blotting System (Invitrogen) following the manufacturer's instructions. Membrane was blocked with 3% BSA overnight at 4°C and then incubated with primary antibodies for 1 hour at room temperature. Antibody against N-terminus MYB was purchased from Abcam (EP769Y). After washed with PBS, the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody for 45 minutes. Bands were visualized with Chemiluminescence (Pierce).

In total 7.5 μg of anti-MYB antibodies (EP769Y, Abcam) were conjugated to 1 mg M-270 Epoxy-coated magnetic beads (Invitrogen) according to manufacturer’s instructions. In total 2 × 10⁷ MV-411 cells were collected and washed in cold PBS. Washed cell pellets were resuspended in 2 ml cold lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 0.5% Triton X-100, 10% glycerol supplemented with protease inhibitors described above) and incubated on ice for 10 min. Cells centrifuged for 5 min at 2000 × g at 4 °C. Supernatant was clarified by centrifugation for 15 min at 18,000 × g at 4 °C. Cleared lysate was added to 1 mg beads, and MYBMIM was added to a final concentration of 20 μM. Immunoprecipitation proceeded for 3 h at 4 °C with rotation. Beads were washed with 1 ml cold lysis buffer twice. Proteins were eluted in 30 μl EB buffer (Invitrogen) for 5 min at room temperature with agitation, and eluate was neutralized with 2 μl 1 M Tris pH 11. Samples were prepared for western blot by addition of Laemmli buffer with 50 mM DTT and incubation at 95 °C for 5 min. The presence of MYB and CBP/P300 was identified by western blot as described.

For MYB immunoprecipitations, 7.5 μg of anti-MYB antibodies (EP769Y, Abcam) were conjugated to 1 mg of M-270 Epoxy-coated magnetic beads (Invitrogen) according to manufacturer’s instructions. For CBP immunoprecipitations, 50 μL of each of Protein A and Protein G Dynabeads (Invitrogen) were combined and washed in 1 mL of PBS with 0.5% BSA. Fifteen μg of anti-CBP antibodies were added to Protein A/G beads in 1 mL PBS with 0.5% BSA and incubated for 1 hr at room temperature with rotation. Beads were then washed with 1 mL PBS with 0.5% BSA and beads were resuspended in a final volume of 100 μL of PBS with 0.5% BSA. One hundred million cells were used per immunoprecipitation. For displacement assays, cells were treated with 10 μM peptides as indicated for 1 hr at 37°C in complete RPMI media prior to nuclear isolation. Immunoprecipitations were carried out using 100 μL of respective antibody-bead slurry per immunoprecipitation overnight at 4°C with rotation. Beads were washed three times with 1 mL of cold lysis buffer. Proteins were eluted in 40 μL of 0.2 M glycine pH 3 for 30 min on a ThermoMixer (Eppendorf) at 900 rpm at room temperature. Eluates were neutralized with 8 μL of 1 M Tris, pH 11. Samples were prepared for western blot by addition of western blot sample buffer described above together with 2.5 µL of 1M DTT and incubated at 95°C for 5 min.