Figure 3
From: Biophysical and functional study of CRL5Ozz, a muscle specific ubiquitin ligase complex
Analysis of CRL5Ozz by glycerol gradient ultracentrifugation microfractionation. (a) Sedimentation profiles of the individual Ozz–EloBC and Cul5–Rbx1 subcomplexes as well as the 1:1 mixture of the two (7.5 μg Ozz–EloBC and 7.5 μg Cul5–Rbx1) were obtained by densitometric measurement of band intensity of eluted proteins after glycerol gradient ultracentrifugation. Aliquots of the first 18 fractions of the total 25 fractions collected were analyzed on SDS–polyacrylamide gel stained with SYPRO Ruby. The Ozz–EloBC (~ 60 kDa, upper panel) and Cul5–Rbx1 (~ 100 kDa, middle panel) subcomplexes eluted in fractions corresponding to their calculated molecular weight. The assembled CRL5Ozz elution pattern shifted to fractions corresponding to its monomeric molecular weight (~ 160 kDa, lower panel). (b) 15 μg of a CRL5Ozz complex and 15 μg of albumin were mixed and run together on a glycerol gradient. The sedimentation profile of CRL5Ozz was not altered by the presence of albumin. (c) Sedimentation profile of Apoferritin (~ 443 kDa), β-amylase (~ 200 kDa) and albumin (~ 60 kDa) protein markers.
