CRBN Immunoprecipitation and Immunoblotting Protocol

CRBN immunoprecipitations were performed as previously described (11 (link)). Proteins precipitated were eluted in 4× Laemmli buffer with 2-mercaptoethanol and used for immunoblotting. For immunoblotting, cells were lysed in 2× Laemmli buffer with 2-mercaptoethanol, and the lysates were run on an SDS-PAGE electrophoresis, followed by protein transfer. The primary antibodies used for immunoblotting include CK1α (Abcam); GAPDH, β-catenin, phosphorylated β-cat S45, and hemagglutinin (Cell Signaling Technology); heat shock protein 90 (HSP90; Santa Cruz Biotechnology); FLAG (Sigma); CRBN (Novus Biologicals); and α-tubulin (EMD Millipore). The secondary antibodies used were horseradish peroxidase–conjugated donkey antimouse or anti-rabbit (Jackson ImmunoResearch). Immunoblots were developed using X-ray films. Chemiluminescence of immunoblots was analyzed by ImageJ software (National Institutes of Health).