Figure 2
From: Biophysical and functional study of CRL5Ozz, a muscle specific ubiquitin ligase complex
Analysis of CRL5Ozz assembly by gel filtration chromatography and coimmunoprecipitation. (a) Affinity purified Ozz–EloBC (~ 60 kDa), Cul5–Rbx1 (~ 100 kDa) and the mixture of the two subcomplexes were visualized on SDS‐polyacrylamide gels stained with SYPRO Ruby. (b) CRL5Ozz chromatography profile showing the elution volume and the fractions corresponding to the three major peaks (Peak 1, Peak 2 and Peak 3). (c) Superose 6 10/300 size exclusion chromatography markers: thyroglobulin, 669 kDa; apoferritin, 443 kDa; β-amylase, 200 kDa; carbonic anhydrase, 29 kDa. (d) The 1:1 mixture was loaded onto a gel filtration chromatography column. Fractions from Peak 1, Peak 2 and Peak 3 were pooled, concentrated and their protein content visualized on SDS‐polyacrylamide gels stained with SYPRO Ruby. All 5 components of CRL5Ozz eluted together mainly in Peak 2 (RV 14.17 = ~ 340 kDa), corresponding to 1D1–1E1 fractions. (e) Purified Ozz–EloBC and Cul5–Rbx1 subcomplexes were mixed in a 1:1 ratio and subjected to immunoprecipitation using either anti-EloC or anti-Rbx1 antibodies. Immunoprecipitated samples were analyzed on SDS–polyacrylamide gels stained with SYPRO Ruby. Both antibodies co-immunoprecipitated all CRL5Ozz components, confirming the formation of the assembled complex by mixing the two subcomplexes in vitro.
