Inverse correlation between endogenous ZBP-89 protein and CD11b mRNA during differentiation of monocytes/macrophages.

(A) Morphology of human monocytes during differentiation and maturation into macrophages is shown at day 1 (left panel) and day 12 (right panel). Representative fields are shown using light microscopy (original magnification, × 40). (B) Real-time RT-PCR analysis ofCD11b during monocyte/macrophage differentiation. Total RNA isolated from monocytes/macrophages differentiated in vitro for 1 or 12 days were reverse transcribed to make cDNA. Quantitative PCR was carried out using the TaqMan system and an ABI Prism 7700 instrument as described in “Materials and methods.” The copy number of CD11b is normalized to the copy number of GAPDH and expressed as CD11b/GAPDH. The CD11b/GAPDH ratio in monocytes at day 1 was given a value of 1, and the ratio at day 12 is shown as a relative value to 1. Each histogram represents the mean ± SD of 3 independent experiments. (C) Western blot analysis showing ZBP-89 protein expression during differentiation of U937 cells and monocytes/macrophages. Five micrograms of nuclear extract from U937 cells treated with PMA for 0 hours (lane 1) or 72 hours (lane 2), and 30 μg total protein lysate from monocytes/macrophages differentiated for 1 (lane 3) or 12 days (lane 4) were resolved by 6% SDS-PAGE. Proteins were transferred to a nitrocellulose membrane and detected with an anti–ZBP-89 antibody followed by chemiluminescence. ZBP-89 (arrow) migrates as a double band with apparent mobility of about 120 kDa, consistent with similar analyses by other investigators30,34 60 MW indicates molecular weight markers (Gibco-BRL).