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Fig. 9. Suppression of hVps34 expression does not impede internalization of the EGFR, but slows initial receptor degradation. Control and hVps34 KD cells were stimulated with EGF after overnight incubation with serum-free medium. (A) KD cells were fixed and co-stained with EGFR and LGP85 antibodies before addition of EGF (0 min) and at different time points (30 min, 60 min) after addition of EGF. Arrows indicate vacuoles positive for both the EGFR and LGP85. The brightness of the EGFR image at 60 minutes was enhanced by approximately 50% to compensate for its weaker EGFR immunofluorescence compared with the brightness of the image at 30 minutes. Bar, 10 µm. (B) To measure EGFR degradation, triplicate cultures of control and KD cells were harvested at the indicated times after the addition of EGF and subjected to immunoblot analysis for total EGFR. The graph illustrates the data (mean±s.e.m.) generated from densitometer scans of ECL signals on film.
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