Figure 8a

Cleavage of Ubiquitin-conjugates in the white muscle with ubiquitylprotein-Isopeptidase. Figure 8a shows the cleavage of ubiquitin-conjugates of the extract and APFII after anion-exchange chromatography from the white muscle (batch 2-7). The incubation batches contained with a final concentration of 5 μg/ml leupeptine and depending on batch 5 mM iodacetamide. All batches were incubated for 60 min in 37°C in a waterbath. 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). Lane 1: 8.5 μg ubiquitin-calmodulin conjugates (Order 1-3) (control). Lane 2: 200 μg white muscle extract, 8.5 μg ubiquitin-calmodulin conjugates (Order 1-3) and 25 μg ubiquitylprotein-isopeptidase Lane 3: 200 μg white muscle extract and 25 μg ubiquitylprotein-isopeptidase. Lane 4: 200 μg white muscle extract and with a final concentration of 5 mM iodacetamide. Lane 5: 200 μg white muscle run-through, 8.5 μg ubiquitin-calmodulin conjugates (Order 1-3) and 25 μg ubiquitylprotein-isopeptidase. Lane 6 200 μg white muscle run-through and 25 μg ubiquitylprotein-isopeptidase. Lane 7: 200 μg white muscle run-through (control) and 5 mM iodacetamide.