Heat shock protein 90 hsp90

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).

Whole cell extracts (WCE) were prepared by freezing the cell pellet overnight at −80°C. The pellet was then resuspended in 3 volumes of WCE buffer (20 mM HEPES, 0.42 M NaCl, 0.2 M EDTA, and 25% glycerol; pH 7.4) plus protease inhibitor cocktail and incubated on ice for ten min followed by 100,000 ×g centrifugation at 4°C. Protein samples were resolved by SDS polyacrylamide gel electrophoresis and electrophoretically transferred to Immobilon-FL membranes. Membranes were blocked at room temperature for 1 hour in TBS [TBS; 10 mM Tris-HCl (pH 7.4) and 150 mM NaCl] containing 3% BSA. Subsequently, the membrane was incubated overnight at 4°C with antibodies to PPARγ (Santa Cruz, 7273), C/EBPα (Santa Cruz, 365318), or heat-shock protein 90 (HSP90) (Santa Cruz, 13119) (Santa Cruz Biotechnology, Dallas, Texas). After three washes in TBST (TBS plus 0.1% Tween 20), the membrane was incubated with an infrared anti-rabbit (IRDye 800, green) or anti-mouse (IRDye 680, red) secondary antibody labeled with IRDye infrared dye (LI-COR Biosciences) for 2 hours at 4°C. Immunoreactivity was visualized and quantified by infrared scanning in the Odyssey system (LI-COR Biosciences).