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Fig. 6. Peptide-mediated downregulation of TNF-R1 but not Fas. (A) HeLa cells were incubated with increasing concentrations of Antp, R9 and Tat for 30 minutes at 37°C. (B) HeLa cells were incubated either with medium alone or with medium containing the TACE inhibitor TAPI-1 (20 µM) for 1 hour at 37°C, then washed and incubated with increasing concentrations of Antp for a further 30 minutes in the presence or absence of inhibitor. (A and B) Cells were harvested with trypsin/EDTA and incubated with the TNF-R1-specific antibody H398 (5 µg/ml) for 1 hour at 4°C. (C) HeLa cells were incubated with increasing concentrations of Antp or R9 for 30 minutes at 37°C, washed, harvested with trypsin/EDTA and incubated with monoclonal anti-CD95 (Fas) antibody (5 µg/ml) for 1 hour at 4°C. (A-C) Cells were washed and incubated with a secondary antibody (Cy5-labelled, goat anti-mouse, 1:500) for 30 minutes at 4°C. For the determination of residual receptor on the cell surface the mean fluorescence in the untreated groups served as a reference (100%). The error bars represent the standard deviation of the mean of three independent experiments.
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