Plasma membrane sequestration of Apaf-1 in BL cells: mobilization by lipid raft–disrupting agents. (A) Raji cells were fixed and stained with anti–Apaf-1 antibodies followed by Alexa 488–conjugated secondary antibodies (green). Simultaneous staining of mitochondria (MitoTracker Red), Golgi (WGA-Texas Red), and nuclei (Hoechst 33342; blue) was performed, and merged confocal images are shown. The same pattern of Apaf-1 distribution was observed in 95 of 100 cells. (B) Merged image of 10 consecutive cross-sections of 1 representative Jurkat cell labeled with Apaf-1–specific antibodies. (C) Representative cross-sections of Apaf-1–labeled Raji cells are displayed in the large panels, whereas several optical sections of each cell were merged in the small panels to generate images in the x-z and y-z axes, respectively. Note the overlay between Apaf-1 (green) and the lipid raft marker, CTB (red). (D) Cytosplasmic redistribution of Apaf-1 upon disruption of the actin cytoskeleton or lipid rafts. Raji cells were incubated for 1 hour in medium alone or in the presence of MCD or CB. Cells were subsequently fixed and stained for Apaf-1 (green) and for lipid rafts, using Alexa 555–conjugated CTB (red). (E) Jurkat cells are labeled as in panel D. No apparent changes in the pattern of Apaf-1 distribution were observed upon incubation with MCD. Original magnification of all panels was × 40.