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. Author manuscript; available in PMC: 2010 Jun 10.
Published in final edited form as: Oncogene. 2009 Dec 10;28(49):4317–4325. doi: 10.1038/onc.2009.287

Figure 2. D1-K269R is resistant to SCFFbx4/αB-crystallin ubiquitylation in vitro.

Figure 2

A. SCFFbx4/αB-crystallin complexes were assembled in SF9 cells, purified with M2 agarose and used to ubiquitylate recombinant GST-D1 or GST-D1-K269R. Reaction mixtures were separated by SDS-PAGE, followed by immunoblot with cyclin D1 antibody. B. D1/K4 and D1-K269R/K4 complexes were purified form SF9 cells using M2 agarose and used for in vitro ubiquitylation reaction as described in A. C. SCFFbx4/αB-crystallin complexes were expressed in SF9 cells, purified with M2 agarose and mixed with GST-WT cyclin D1, D1-T286A or D1-K269R for binding analysis. Complexes were separated by SDS-PAGE, followed by immunoblot with cyclin D1 and Fbx4 antibodies.