Figure 5. IgE/FcεRI complexes are retained within lipid raft–enriched domains of BMMCs following exposure to pAgs, but not to sAgs.

(A) Association of IgE/FcεRI complexes (red) with lipid rafts (green) at the indicated time points following exposure to sAgs or pAgs. Shown in blue are pAgs. Arrows denote examples of colocalization between IgE/FcεRI complexes and lipid raft–enriched regions. Arrowheads denote examples in which IgE/FcεRI complexes have separated from lipid raft–enriched compartments and no colocalization is evident. Scale bar: 5 μm. (B) Association of FcεRI-γ chain with lipid raft fractions in sensitized BMMCs before and at various time points after exposure to sAgs or pAgs. Cell fractions were obtained after sucrose gradient centrifugation and probed for FcεRI-γ chain on a Western blot. Distribution of FcεRI-γ chain in cell fractions prior to addition of allergen is also shown. Based on the location of flotillin-1, fractions 4 and 5 are defined as the lipid raft fractions. (C) Sucrose gradient fractions 4 and 5 were pooled and subjected to Western blot to detect tyrosine phosphorylation within lipid fractions of BMMCs at various time points after exposure to sAgs or pAgs. Arrows denote phosphorylated bands that appeared more intense in pAg- relative to sAg-stimulated cells. Arrowhead denotes a band for which differences between sAg- and pAg-stimulated cells was not marked. Images are representative of 3 separate experiments.