Figure 4.

mAb 15308 reveals a 40 kDa protein associated with apoptotic cell-derived microvesicles. (a) SDS-PAGE and immunoblots of lysate from MUTU I BL cells fractionated by sequential centrifugation into 1000 × g (N), 27 000 × g (P1), 100 000 × g (P2) pellets and remaining supernatant (S). (b) Flow cytometric analysis of mAb 15308 reactivity with MV produced by MUTU I BL cells with (left panel) and without (right) induction of apoptosis by UV irradiation for 16 h. Black histogram indicates isotype control binding. (c) Immunoblotting of mAb 15308 reactivity with MVs produced by MUTU I BL cells after induction of apoptosis by UV irradiation for 16 h. (d) Quantification by protein assay of MVs liberated by 10 × 10⁶ BL2 cells and Bcl-2-overexpressing BL2 cells (BL2-Bcl-2) following induction of apoptosis by staurosporine. One-tailed unpaired Mann–Whitney test; *P≤0.05. By 4 h, >70% of staurosporine-treated BL2 cells bound AxV, compared with 5–25% BL2-Bcl-2 cells. (e) Flow cytometric analyses of mAb 15308-stained MVs isolated from BL2 and BL2-Bcl-2 cells following apoptosis induction by staurosporine for 3 h. Histograms of representative samples of MVs isolated from 3 h staurosporine-treated BL2 (left panel) and BL2-Bcl-2 (middle) cells are shown. Percentages of 15308-positive MVs are indicated on the histograms and mean percentages±S.E.M (n=3 independent experiments for each cell type) are shown (right). One-tailed unpaired Mann–Whitney test; *P≤0.05