Figure 6a

Cleavage of Ubiquitin-conjugates in the heart muscle with ubiquitylprotein-isopeptidase. Figure 6a shows the cleavage of ubiquitin-conjugates of the extract heart muscle pass-through (batch 2-7). All batches were incubated for 60 min in 37°C in a water bath. The reaction was stopped by adding trichloracetic acid (final concentration 5% w/v TCA, 20 min 0°C). After centrifugation for 5 min in an Eppendorf centrifuge 5415 (Eppendorf, Hamburg) at 16000 × g the pellet was solved in sample buffer for Laemmli system and was blotted on PVDF-membrane (see materials). Lane1: 8.5 μg ubiquityl-calmodulin conjugates (Order 1-3). Lane 2: 8.5 μg ubiquityl-calmodulin conjugates (Order 1-3) and 12.5 μg ubiquitylprotein-isopeptidase. Lane 3: 200 μg heart muscle run-through + 8.5 μg ubiquityl-calmodulin conjugates (Order 1-3) and 12.5 μg ubiquitylprotein-isopeptidase. Lane 4: 200 μg heart muscle run-through + 8.5 μg ubiquityl-calmodulin conjugates (Order 1-3) and 25 μg ubiquitylprotein-isopeptidase. Lane 5: 200 μg heart muscle run-through and 25 μg ubiquitylprotein-isopeptidase. Lane 6: 200 μg heart muscle run-through, 25 μg ubiquitylprotein-isopeptidase and 5 mM iodacetamide. Lane 7: 200 μg heart muscle run-through (control).