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Fig. 3. High levels of LAMP1-GFP overexpression elicit aggregation of late endocytic compartments. Immortalized skin fibroblasts from wild-type (A-H) and pale ear (I-L) mice were transfected with plasmids encoding LAMP1 fused to either GFP or its non-dimerizing mutant form (mGFP). One day after transfection, cells were fixed/permeabilized and stained with the 1D4B antibody to mouse Lamp1 (B,D,H,J). In some experiments, cells were first allowed to internalize Texas-Red-conjugated dextran (F,L), transfected with the LAMP1-GFP-encoding plasmid, cultured for one day in medium without dextran, and then fixed and processed for fluorescence microscopy. Arrows indicate cells expressing high levels of LAMP1-GFP or LAMP-mGFP, as judged by their GFP fluorescence signal (A,C,E,G,I,K). Asterisks in A and B denote a transfected cell expressing LAMP1-GFP levels comparable with those of the cells shown in Fig. 2. Bar, 20 µm. The inset in F shows a high-resolution view of one of the dextran clusters as obtained by confocal microscopy, suggesting that the dextran-positive compartments have aggregated but not fused with each other.
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