|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 3. Changes in the lysosomal degradation of LAMP-2A with age. (A) Half-life of LAMP-2A in cultured primary mouse fibroblasts. Early [population doubling level (PDL) 4-6] and late (PDL 12-15) passage primary mouse fibroblasts were labeled with [35S]methionine:cysteine mixture and at the indicated times cells were lysed and subjected to immunoprecipitation with the antibody against the LAMP-2A cytosolic tail. Immunoprecipitates at different times from cells maintained in the presence (serum+) or absence (serum–) of serum were subjected to SDS-PAGE and fluorography, which were quantified by exposure to a PhosphorImager screen. Values are expressed as a percentage of radiolabeled LAMP-2A at time 0 and are mean of three different experiments. The best exponential decay curve was calculated by linear regression analysis and the half-life of LAMP-2A was calculated by the formula t1/2=ln2/degradation rate. (B) Degradation of LAMP-2A in lysosomes. Liver lysosomes from 4- and 22-month-old rats were incubated at 37°C in an isotonic medium. At the indicated times lysosomes were pelleted and membranes and matrices were isolated after hypotonic shock by centrifugation. Samples were divided into two halves and subjected to SDS-PAGE and immunoblot with the antibody that recognizes the cytosolic tail of LAMP-2A or an antibody against the luminal region of LAMP-2, common to all spliced variants (shown here). The graph shows the changes in the amount of cytosolic tail at different times expressed as percentage of the amount present before the incubation. Values are mean ± s.e.m. of three different experiments. Filled arrowhead indicates the truncated form of LAMP-2A generated by cleavage of the protein at the lysosomal membrane, while open arrowhead indicates an unusual lower molecular weight LAMP-2A detected only in the lumen of lysosomes from the oldest animals.
|
