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Fig. 8. HA-FYVE2-TOM1 chimera recruits clathrin to endosomes while mutation of CB-I and CB-II reduces this activity. (A) Schematic diagram of the chimeric constructs used in the experiment. Mutated residues are indicated in italics and deletions are indicated by a gap. (B) HA-FYVE2-TOM1 recruits clathrin to endosomes whereas the chimeric CB-I and CB-II single or double mutants are significantly less effective. A431 cells were transfected with each of the constructs depicted in A and processed for immunofluorescence analysis by fixing with paraformaldehyde followed by permeabilization with 0.05% saponin. The cells were stained with anti-HA (left panels and green in merged images) and anti-clathrin heavy chain (middle panels and red in merged images) and analyzed by confocal microscopy. Merged images are shown in the right panels where yellow represents areas of colocalization. CB, CB-I and CB-II, clathrin-binding sites; EBD, endofin-binding domain; GAT, GGAs and TOM1 homology; VHS, VPS-27, Hrs and STAM. Bar, 10 µm.
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