Fig. 9.
Inhibition of lipid raft-dependent endocytosis blocks βIP generation and IL-5-mediated signaling. (A) Cytokine-starved TF1 cells (1×106 cells/tube) were either left untreated (upper left panel) or treated with 2.5 μg/ml filipin (upper right panel) or 25 μg/ml NYS (lower panel) for 30 min to disrupt lipid raft microdomains. βc cell surface expression was measured by flow cytometry before (–IL-5, shaded histograms) and after 30′ IL-5 stimulation (+IL-5, solid black line). The hatched line represents cells labeled with an isotype-matched control antibody [C]. Mean fluorescence intensities (MFI) are plotted and sem are listed in the text. Untreated n = 5; FP n = 4. (B) TF1 cells were pretreated the same as in Fig. 9A, followed by an IL-5 (10 ng/ml) time course assay for 0, 30, and 60 min. WCLs were prepared, IP with anti-βc mAb (S-16), and serially IBed with the listed antibodies in the figure. Note how both βIP bands (βIP 1 and 2) are greatly reduced, with the simultaneous accumulation of ubiquitinated βc receptors in the presence of filipin (even-numbered lanes). Also, note how CO-IPs of 5Rα and JAK2 are blocked in the filipin-treated lanes (even-numbered lanes).