Figure 6
From: Biophysical and functional study of CRL5Ozz, a muscle specific ubiquitin ligase complex
Glycerol gradient ultracentrifugation microfractionation analysis of CRL5Ozz and its substrates. Assembled CRL5Ozz mixed with purified preparations of (a) GST- full length β-catenin, (b) GST-MyHCemb fragment (1041–1941 a.a.) and (c) GST-full length Alix was fractionated from a post-ultracentrifuged glycerol gradient (8 h). Aliquots of each fraction were separated on SDS–polyacrylamide gels and their protein content visualized on gels stained with SYPRO Ruby. The densitometric measurement of band intensity of the proteins in each fraction showed a shift to a higher molecular weight when CRL5Ozz was mixed with either of its substrates, compared to the molecular weights of CRL5Ozz or its individual substrates: CRL5Ozz–β-catenin (~ 272 kDa), CRL5Ozz–MyHCemb fragment (1041–1941 a.a.) (~ 280 kDa) or CRL5Ozz–Alix (~ 282 kDa). (d) Sedimentation analysis of Alix mixed with albumin as internal control. The fractions were loaded on an SDS–polyacrylamide gel and stained SYPRO Ruby. Alix and albumin were visible in fractions 2–12 and the profile of either protein was not altered in presence of the other.
