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Fig. 2. Age-related changes in the dynamic distribution of LAMP-2A in lysosomes. (A) Distribution of LAMP-2A between lysosomal membrane and matrix. Membranes (MB) and matrices (MTX) from lysosomes isolated from livers of 4-, 12- and 22-month-old rats were subjected to SDS-PAGE and immunoblot against LAMP-2A. The percentage of total lysosomal LAMP-2A present in each compartment was calculated by densitometry of the immunoblots. Values are mean + s.e.m. of four to five different experiments. (B) Mobilization of LAMP-2A between the lysosomal membrane and matrix. Intact lysosomes from livers of 4-, 12- and 22-month-old rats were incubated in the presence of GAPDH (25 µg) for 15 minutes at 37°C and then the substrate was rapidly removed by centrifugation (arrows) and lysosomes were incubated in a substrate-free isotonic medium. At the indicated times, lysosomal membranes were isolated and subjected to immunoblot against LAMP-2A. Values are expressed as the percentage of LAMP-2A present at the lysosomal membrane at the beginning of the incubation and are the mean + s.e.m. of three different experiments. Right panels show representative immunoblots. (C) Rates of LAMP-2A internalization (left) and retrieval back to the lysosomal membrane after removal of GAPDH (right) were calculated from the data in B. Values are expressed as changes in the percentage of membrane LAMP-2A per minute. (D) Mobilization of recombinant LAMP-2A into lysosomal membranes. Purified recombinant HA-tagged LAMP-2A was placed into resealed lysosomal membranes isolated from 4-, 12- and 22-month-old rats as described under Materials and Methods. Vesicles were incubated at 37°C for 15 minutes and the amount of HA-LAMP-2A associated with the lysosomal membrane was quantified by immunoblot against HA of the membranes isolated by hypotonic shock and centrifugation (protease protection assay was used to determine that all the HA-LAMP-2A associated with the membrane had a cytosolic exposed C-terminus). Values are expressed as percentage of the total LAMP-2A incorporated in each group of resealed membranes and are mean + s.e.m. of three different experiments. Significant differences were observed compared with values from 4-month-old rats (*P<0.05; **P<0.01).
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