Fig. 1. Characterization of lymHsc70. (a) Two-dimensional gel electrophoresis of lysosomal Hsc70. The numbers at the top refer to isoelectric points. Top, untreated IMR-90 human fibroblast lysosomes (100 µg protein); middle, lysosomes treated with trypsin (100 µg); bottom, untreated purified lysosomal membranes (100 µg). Blots were probed with mAb 13D3 (anti-Hsc70) and developed with chemiluminescense (see Materials and Methods). (b) Lysosomal membranes (30 ug) were treated with 0.5 M NaCl (lane 1), 0.1 M Na2CO3, pH 11.0 (lane 2), or 5.0 M urea (lane 3). Samples were analyzed by SDS-PAGE and transferred to Immobilon-P membranes. Blots were probed with anti-Hsc70 and anti-Lamp2a antibodies and developed with chemiluminescense (see Materials and Methods). (c) Purified lysosomes (100 µg) were incubated as indicated without additions, with ADP/Mg2+, the protein substrate, RNase A, or RNase A plus ADP/Mg2+. Samples were incubated or not for 20 minutes with proteinase K (10 µg/ml). Samples were separated by SDS-PAGE, transferred to Immobilon-P membranes, probed with mAb 13D3, and developed with chemiluminescense (see Materials and Methods). Blots were quantitated by densitometry.
