Fig. 4. Levels of chaperones and cochaperones on the lysosomal membranes and in the cytosol. (a) Lysosomes were from cells maintained under serum-supplemented (+S) or serum-starved (-S) conditions for 18 hours. Lysosomal membrane proteins (30 µg) were separated by SDS-PAGE, transferred to Immobilon-P membranes, probed with corresponding antibodies, developed with chemiluminescense (see Materials and Methods), and quantified by densitometry. Values shown (±s.d.) are averages of three separate experiments (top). Cytosolic proteins (15 µg) were analyzed similarly (bottom). (b) Purified lysosomes (50 µg) were incubated at different times with an ATP-regenerating system without an added protein substrate. After incubation for increasing times, lysosomal membranes were purified and analyzed by SDS-PAGE and western blot. Levels of the indicated proteins were determined by densitometry and expressed as percentage of the value at the beginning of the experiment.
