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Table 2.

Density of lgp120 immunogold labelling over endosomes and lysosomes

Tf-HRP endosomes
 
Lamin A siRNA
 
TSG101 siRNA
 
Membrane (μm)   14.07   16.11  
Total lgp120 particles   9   17  
Particles per μm membrane
 
0.6
 
1.1
 
Lysosomes
 
Lamin A siRNA
 
TSG101 siRNA
 
Membrane (μm)   14.32   16.11  
Total lgp120 particles   237   268  
Particles per μm membrane   16.6   16.6  
Tf-HRP endosomes
 
Lamin A siRNA
 
TSG101 siRNA
 
Membrane (μm)   14.07   16.11  
Total lgp120 particles   9   17  
Particles per μm membrane
 
0.6
 
1.1
 
Lysosomes
 
Lamin A siRNA
 
TSG101 siRNA
 
Membrane (μm)   14.32   16.11  
Total lgp120 particles   237   268  
Particles per μm membrane   16.6   16.6  

TSG101-depleted and lamin A-depleted cells were incubated with EGF and 108 anti-EGFR 10 nm gold for 30 minutes at 4°C, washed, and incubated at 37°C for a further 1 hour, with TfHRP added for the final 30 minutes. The cells were then post-fix labelled with 5 nm gold for lgp120. Measurements were made of membrane length of Tf-HRP-containing endosomes and of lysosomes (scored by morphology; heterogeneous content, and single perimeter membrane containing at least one lgp120 gold particle) in control cells (from 6 cells) and of Tf-HRP-containing multicisternal endosomes and of lysosomes from TSG101-depleted cells (6 cells; only membrane exposed to cytosol was measured, based on the assumption that antibody would be excluded from intercisternal areas). Lgp120 gold particles on measured membrane were counted.

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