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. 2011 Aug 3;286(39):34060–34070. doi: 10.1074/jbc.M111.273045

FIGURE 4.

FIGURE 4.

Isolation and identification of the hDCNL1 E3. A, HeLa extracts catalyze the mono-ubiquitination of hDCNL1. Reaction was carried out and analyzed as described under under “Experimental Procedures.” B, flow chart outlines a strategy for the isolation and identification of hDCNL1 E3. See “Experimental Procedures” for details. C, molecular weight estimation of the hDCNL1 E3. Top, gel filtration analysis. A Stokes radius standard curve, generated using molecular size markers, is shown. The position of the hDCNL1 E3 is marked, which corresponds to a Stokes radius of 5.58 nm. Bottom, glycerol gradient analysis. A sedimentation S value standard curve, generated using molecular size markers, is shown. The position of the hDCNL1 E3 is marked, which corresponds to an S value of 5.47 S. D, Nedd4-1 is co-migrating with the hDCNL1 mono-ubiquitination activity. Aliquots (3 μl) of the indicated glycerol gradient fractions were analyzed by the hDCNL1 mono-ubiquitination assay. The protein peak of Nedd4-1 is revealed by immunoblot analysis with antibodies specific for Nedd4-1. Load: an aliquot of the starting material for glycerol gradient sedimentation. Thy: thyroglobulin (Mr = 670,000); Cat: catalase (Mr = 240,000); BSA: bovine serum albumin (Mr = 66,000).