Association of growth factor receptors with and integrity of lipid rafts in myeloma cell apoptosis. Immunoprecipitation (IP) using antibody specific to (A) caveolin-1 (Cav-1), (B) IL-6R gp130, and (C) IGF-IRβ in myeloma cells treated with cytokines (Cyto; IL-6 or IGF-I) or cytokines together with mAb D1 (Cyto + D1), followed by Western blotting analysis (WB) using antibodies against gp130, IGF-IRβ, MHC class I (W6/32), or caveolin-1. (A-C) Expression of caveolin-1, gp130, and IGF-IRβ, respectively, serve as loading controls. Results obtained with D1 mAb on MM.1S from 1 representative experiment of 4 performed are shown. Similar results were obtained with other tumor cell lines. (D) To deplete cholesterol and disrupt lipid rafts, cells were preincubated with MCD (5 mM; titrated in preliminary experiments) for 30 minutes, washed, and incubated further with β₂M-specific mAb D1 (50 μg/mL) or dexamethasone (10 μM) in the presence or absence of IL-6 (10 ng/mL). Percentage of apoptotic cells was measured at 48 hours by Annexin-V–binding assay. Results from 4 experiments performed are shown. Similar results were obtained with anti-β₂M mAb E6. Error bars indicate SD.