I3C regulation of nucleostemin–MDM2 and p53-MDM2 protein interactions in well-established human breast cancer cell lines. SKBR3 (A), MCF-7 (B) and MDA-MB-231 (C) human breast cancer cells were treated with or without 200 μM I3C for 48 hours. Total cell extracts were immunoprecipitated with either MDM2 (top panels for each cell line) or nucleostemin (lower panels for each line) antibodies. As a control, non-immune antibodies (IgG) and samples not immunoprecipitated (No IP) were used. All extracts were electrophoretically fractionated and probed by Western blot analysis using antibodies specific to p53 (top panels) or with antibodies specific to serine-166 phosphorylated MDM2 or total MDM2 (lower panels). The levels of actin protein remaining in the cell extracts after the immunoprecipitations were used as gel-loading controls in each experiment. I3C, indole-3-carbinol; IgG, immunoglobulin G; IP, immunoprecipitated; MDM2, murine double mutant 2; NS, nucleostemin.