Figure 5
Western blot assay of ubiquitylated proteins in fractions obtained from rabbit reticulocytes. Reticulocytes were prepared in presence of 10 mM glucose, 145 mM NaCl, 5 mM KCl, 1 mM Ca2+, 0.15 mM MgCl2, 0.1 mM EGTA, 10 mM HEPES, pH 7.4 and lysed in presence of 10 mM iodacetamide. 250 ml lysate (63.22 mg/ml) was prepared as described in Material. Reaction mixtures for uCaM-isopeptidase (lane 2 and 4) contained in a total volume of 230.8 μl 50 mM Tris/HCl, 5 mM Mg acetate, 50 μg enriched uCaM-isopeptidase fraction. Incubation was stopped after 60 min. by adding of TCA at final concentration of 5%. The pellets were neutralized with 60 μl Laemmli sample buffer, heated to 96°C for 10 min and then subjected for SDS-PAGE. Lane 1: 8.5 μg ubiquityl-calmodulin conjugate. Lane 2: 8.5 μg ubiquityl-calmodulin-conjugate + 50 μg ubiquitylprotein-isopeptidase. Lane 3: 100 μg reticulocyte APF II. Lane 4: 100 μg reticulocyte APF II + 50 μg ubiquitylprotein-isopeptidase. Ubiquitylated proteins, first- and second order ubiquityl-calmodulin-conjugates (uCaM I and uCaM II) and free ubiquitin are indicated with arrows.