FIG 4.

Siglec-8 is internalized and shuttled to the cell surface via independent pathways. Eosinophils were pretreated with a panel of inhibitors at the indicated concentrations. Their effects on Siglec-8 endocytosis (A) or Siglec-8 shuttling to the cell surface (B) were assessed at 120 minutes. Siglec-8 shuttling was measured by detecting surface Siglec-8 that was not bound by the unconjugated primary antibody initially. C, Eosinophils were pretreated with the indicated concentrations of sucrose as in Fig 2. Total surface Siglec-8 was divided into an original surface pool and a shuttled pool at 120 minutes and are represented in a stacked column, normalized to total Siglec-8 surface levels at 0 minute. D, Stacked columns of the original surface pool of Siglec-8 and the shuttled pool at various durations of incubation at 37°C in the presence or absence of LY294002 and BIM (both at 10 μM). Data represent the means ± SDs of 3 or more independent experiments. *P < .05, **P < .01, ***P < .001, and ****P < .0001 relative to untreated eosinophils.