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Volume 5 issue 9 September 2004
Lysosome associated membrane protein 1 (Lamp1) traffics directly from the TGN to early endosomes.
Neil R Cook, Paula E Row & Howard W Davidson
ONLINE SUPPLEMENTAL MATERIAL
As a control for surface delivery we used the YAL Y/A chimera (Figure 2K). After a 10 min pulse with 35S-sulfate, chase at 37°C, and incubation with anti-Av on ice, a small population of sulfated YAL Y/A could be captured after 15 min of chase (lower panel). The amount captured continued to increase over the rest of the chase. In contrast at no chase time was this possible for the chimera containing the wild-type cytoplasmic tail (upper panel and unpublished data). This indicates that like its parent molecule, YAL appears to be delivered to the endocytic directly without prior exposure at the cell surface.
Supplemental Figure. Surface expression of the YAL and YAL Y/A chimeras . Hela D3 cells (upper panel) or B2 cells (lower panel) were treated with 1µM Dex for 24h, then pulse labelled with 35S-sulfate for 10min and chased for times as indicated. At the end of each chase cells were incubated for 2h with anti-Av serum on ice to tag surface molecules. Following extensive washing, cells were lysed and the tagged (surface) molecules ( S ) recovered from the clarified lysates using protein A agarose. The labelled chimera remaining in the lysates ( T ) was determined by the addition of fresh anti-Av serum and protein A agarose. Samples were analysed by SDS-PAGE and phosphorImaging.
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