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Fig. 1. EGF stimulation rapidly induces highly dynamic SNX5-positive tubule formation on newly formed macropinosomes. (A) HEK-GFP-SNX5 cell monolayers were serum starved overnight before being exposed to 100 ng/ml recombinant EGF. The interval between image capture was 10 seconds. EGF was added 1 minute and 40 seconds into the recording (+EGF). Selected frames are presented with the time captured relative to the first frame recorded at the top right (minutes:seconds). The periphery of the cell is marked in yellow. Asterisks indicate the lumen of an individual macropinosome. (B) HEK-GFP-SNX5 cell monolayers were serum starved overnight before being incubated with dextran conjugated to tetramethylrhodamine (dextran-TR) and 100 ng/ml EGF for 3 minutes before fixation with 0.9% PFA at 4°C. SNX5-positive macropinosomes were then analysed by immunofluorescence microscopy. (C) HEK293 cell monolayers were co-transfected with pEYFP-Rabankyrin 5 and pCMU-myc-SNX5 as described in the Materials and Methods, fixed with 0.9% PFA and analysed by indirect immunofluorescence using an anti-myc monoclonal antibody followed by Cy3-conjugated goat anti-mouse IgG. Bars, 10 µm.
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