2.4. Co-Immunoprecipitation

MDA-MB-231 and MCF-7 cells, either as wild type, or transfected with pCMV/MAD-SID, or treated with 5 µM of the MAD-Scr or MAD-SID peptide, were used for the coimmunoprecipitation assays. Treated cells were collected and lysed in a Pierce IP reagent, and 2000 µg of lysates were immunoprecipitated overnight with a primary antibody. The immune complex was immobilized on A/G beads, eluted in a 2X sample buffer and subjected to SDS-PAGE, followed by Western blotting. The amount of Sin3A bound to RARα/RXRα was visualized by chemiluminescence. Mouse IgG were used as a negative control, and a 10% input was used as internal control. For all the blots, including Western blots, the protein bands were quantitated using the ImageJ software. The quantitations of proteins for Western blots were normalized against β-actin, while the coimmunoprecipitated protein (Sin3A) was normalized against inputs (RARα and RXRα).