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Fig. 8. CPP mediate internalization of receptors in a dynamin-dependent but lipid raft-independent manner. (A) HeLadynK44A cells were incubated with or without tetracycline for 48 hours at 37°C, washed and then incubated with medium containing 20 µg/ml Alexa Fluor 633-labelled transferrin for 30 minutes at 37°C. After washing, cells were analysed by confocal microscopy. (B) HeLadynK44A cells were transfected by electroporation with a plasmid coding for human TNF-R2 and incubated for 48 hours at 37°C either with or without tetracycline. TNF-R2 was labelled with a TNF-R2-specific antibody/Zenon Alexa Fluor 647 conjugate. Cells were incubated with Antp (20 µM) for 30 minutes at 37°C, or remained untreated. After washing with medium cells were analysed by multichannel confocal microscopy. (C) EGFR was labelled with Zenon Alexa Fluor 647 conjugate for 30 minutes at 4°C, then cells were washed and incubated with R9 peptide for 30 minutes at 37°C. Scale bar: 20 µm. (D,E) HeLa cells were treated with methyl-ß-cyclodextrin (MßCD; 5 mM) for 30 minutes then incubated with Antp in the presence or absence of the inhibitor for further 30 minutes. Cells were then harvested with trypsin/EDTA, TNF-R1 was immunostained and cells were analysed by flow cytometry. Peptide-associated fluorescence (fluorescein) and Alexa Fluor 647 signal were detected simultaneously.
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