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. 2008 Nov 28;283(48):33211–33220. doi: 10.1074/jbc.M804440200

FIGURE 4.

FIGURE 4.

SCCRO interacts with the unique N terminus of Ubc12∼NEDD8. A, Western blot for Ubc12 after a thioester reaction showing generation of Ubc12∼NEDD8 thioester (top panel, lane 1, upper band). GST (lane 2) and GST-SCCRO (lane 3) pulldown on the same reaction products showing preferential interaction of SCCRO with Ubc12∼NEDD8 despite a large excess of free Ubc12 (top panel, lane 3). Autoradiograph of products from a thioester reaction containing E1, E2, ATP, and 32P-labeled PK-NEDD8 showing Ubc12∼NEDD8 (bottom panel, lane 1, upper band) and free NEDD8 (bottom panel, lane 1, lower band). GST (lane 2) or GST-SCCRO (lane 3) pulldown assays on thioester reaction components showing SCCRO binds to Ubc12∼NEDD8 but not to free NEDD8 (bottom panel, lane 3) B, Western blot showing levels of Ubc12∼NEDD8 and Ubc12 from a thioester reaction supplemented with varying concentrations of free Ubc12 after quenching with EDTA (see “Experimental Procedures”; top panel). The same reactions were subjected to pulldown assays using limiting amounts of GST-SCCRO (70 pm) showing preferential binding to Ubc12∼NEDD8 even in the presence of 20-fold excess of free Ubc12 (bottom panel). C, Western blots for NEDD8 (top panel) and Ubc12 (middle panel) on thioester reactions with and without ATP in lysates from HeLa cells transfected with pCMV or pCMV-SCCRO showing lower levels of Ubc12∼NEDD8 in reactions not containing ATP (compare first and third lanes with the second and fourth lanes). HA immunoprecipitation of the same reaction mix showing preferential binding of SCCRO (in limiting concentrations) to Ubc12∼NEDD8 (bottom panel, third and fourth lanes), even in the presence of a large excess of free Ubc12 (bottom panel, third lane). D, Western blot for HA after thioester reactions on HeLa cell lysates transfected with HA-Ubc12 or HA-Ubc12Δ26 showing equal amounts of NEDD8 thioester formation after 10 and 30 min of incubation, respectively. HA immunoprecipitation on the same reaction products followed by Western blot with the indicated antibody showing SCCRO interacts with full-length Ubc12∼NEDD8 but not with Ubc12Δ26∼NEDD8, whereas ROC1 interacts with both (bottom right).