Coimmunoprecipitation of CRBN

3 × 10⁶ wild-type Hep3B cells were plated onto 15-cm dishes and transfected with 35 μg empty vector or pLX304-ZMYM2, which encodes ZMYM2-V5. Cells were treated with DMSO or 20 μmol/L avadomide in the presence of 10 μmol/L MG-132 for the last 3 hours before harvest. Cells were harvested 48 hours posttransfection and lysed in lysis buffer (as described above) containing 20 μmol/L avadomide or DMSO. Whole-cell extracts were cleared by centrifugation at 13,000 × g at 4°C for 15 minutes, normalized, and incubated with anti–V5-tag magnetic beads (MBL International) at 4°C for 16 hours. After washing the beads three times in wash buffer (150 mmol/L NaCl, 50 mmol/L Tris-HCl pH 8.0, 1% glycerol, 1× Halt Protease, and Phosphatase Inhibitor Cocktail) containing 20 μmol/L avadomide or DMSO, anti-V5 immunoprecipitates were eluted by boiling in Laemmli SDS sample buffer at 95°C for 5 minutes and then subjected to immunoblot analysis for CRBN.