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Fig. 5. Age-related changes in the lipid microdomains at the lysosomal membrane involved in CMA regulation. (A) Lysosomes from livers of fed or 48-hour-starved 4- and 22-month-old rats were extracted with 1% Triton X-114 and then subjected to sucrose gradient centrifugation. Aliquots collected from top to bottom were grouped as the detergent-resistant (DR), intermediate (Int) and detergent-soluble (Sol) fractions, which were then subjected to SDS-PAGE and immunoblotted for LAMP-2A. The graph on the right shows the mean value of the densitometric quantification of the LAMP-2A immunoblots from three experiments. Values are expressed as a percentage of the total LAMP-2A at the lysosomal membrane present in each region. (B) Flotillin-1 (Flot-1) content in lysosomes (Lys) and detergent-resistant regions (DR) isolated and processed as in A from livers of fed 4- and 22-month-old rats. Because Flotillin-1 localizes preferentially in the DR region, levels in the soluble fraction were undetectable. (C) Lysosomal membranes from 4- and 22-month-old rats and their corresponding Triton X-114-resistant (DR) and soluble (Sol) regions were subjected to SDS-PAGE and staining with Sypro Ruby. The densitometric electrophoretic profile of the detergent-resistant regions is shown (bottom). Arrowheads indicate decreased (white) or increased (black) levels compared with 4-month-old rats. (D) Quantification of cholesterol content in homogenates (Top) and lysosomes (bottom) from 4-, 12- and 22-month-old rat livers was carried out by the Amplex Red cholesterol assay. Values are expressed as total units in the fraction and are the mean + s.e.m. of triplicate samples in two different experiments. Significant differences were observed compared with values from 4-month-old rats (*P<0.05, **P<0.01).
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