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Fig. 10. Recycling pathways to the TGN and plasma membrane are disrupted by TSG101 depletion. (A) Lamin A-depleted (panels A-C,G-I) or TSG101-depleted (panels D-F,J-L) HeLa cells were fixed directly (A-F) and stained for Golgin-84 (green; A,D) and TGN46 (red; B,E), or allowed to internalise Alexa Fluor488-labelled EGF for 3 hours at 37°C prior to fixation (G-L), then examined by fluorescence microscopy for EGF (green; G,J) and transferrin receptor (red; H,K). Merged images are shown in panels C,F,I,L. Bar, 10 µm. (B) Upper panel: ¹²⁵I-Tf was bound to lamin A-depleted or TSG101-depleted HeLa cells for 30 minutes at 4°C, after which the cells were washed to remove unbound material and warmed to 37°C in the presence of excess unlabelled Tf for the indicated times. Cells were chilled on ice and washed with a deferoxamine cycle to remove residual surface-bound Tf before being extracted in 1 M NaOH and counted for radioactivity. Results are expressed as the percentage of Tf that remained cell associated relative to that which had been initially bound. Results are the means from two experiments each performed in duplicate ± s.d. Lower panel: results (expressed as cpm ± s.d.) from samples to which ¹²⁵I-Tf was bound at 4°C but not subjected to a deferoxamine wash. (C) Lamin A-depleted (L) or TSG101-depleted (T) HeLa cells were western blotted for TSG101 and TR. (D) Control (upper panel) and TSG101-depleted (lower panel) cells were incubated with Texas red-conjugated Tf for 60 minutes at 4°C followed by 5 minutes at 37°C, then fixed and examined by confocal microscopy. The photomultiplier and image analysis settings were identical in both samples. Bar, 10 µm.

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