Fig. 4. Trafficking of P2X₄ receptors to phagosomes. (A) Primary macrophages were incubated with latex beads or zymosan particles for 30 minutes, followed by a chase for 60 minutes, or incubated for 90 minutes with E. coli expressing GFP. The cells were fixed, permeabilized and stained with antibodies against P2X₄ (green) and LAMP-1 (red). (B) Confocal images of RAW264.7 cells expressing P2X₄-GFP (green) incubated with latex beads for 30 minutes, followed by a 1 hour chase, fixed, permeabilized and stained with antibody against LAMP-1 (red). Bars, 10 µm. (C) Detection of P2X₄ and LAMP-1 in latex bead phagosomes isolated by sucrose density-gradient flotation from bone-marrow-derived macrophages by immunoblotting (`Phag') and dilutions of total cell lysates (`Total').