Fig. 4.

Internalized IL-5Rs colocalize with the lysosomal marker, LAMP-1. Cytokine-starved TF1 cells (upper panels) and eosinophils, which had been cultured in 1 ng/ml human IL-5 for 1 day and cytokine-starved for 1-2 days (lower panels), were processed as described in Figs. 1 and 2 with 250 ng/ml cold Cy3-IL-5 and either left on ice (0 min) or transferred to 37°C for 30 or 60 min. After acid washing, the 30- and 60-min time points only, all cells were fixed, cytospun, and stained with mouse anti-human LAMP-1 mAb (1:50 dilution, BD Biosciences), followed by incubation with 1:500 dilution of donkey anti-mouse IgG-Alexa-647, whose color was changed to green for visualization and colocalization analysis (for TF1 cells), or goat anti-mouse IgG-Alexa-488 for EOS. Nuclei are counterstained with DAPI (blue). Percentage of TF1 cells showing colocalization between Cy3-IL-5 and LAMP-1 was 18%; EOS had 10% colocalization (6+ cells/60; n=2).