Components of the ubiquitin-proteasome pathway compete for surfaces on Rad23 family proteins

Amanda M Goh¹^,5 , Kylie J Walters² , Suzanne Elsasser³ , Rati Verma⁴ , Raymond J Deshaies⁴ , Daniel Finley³ and Peter M Howley¹

¹Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA

²Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota, USA

³Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA

⁴Department of Biology, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California, USA

⁵Institute of Molecular and Cell Biology, Singapore

author email corresponding author email

BMC Biochemistry 2008, 9:4doi:10.1186/1471-2091-9-4


Published:	30 January 2008

Additional files

Additional file 1:

Retention of proteins on the GST-HHR23A~ΔUBL column corresponds to their ability to interact with UBA domains. (A) Purified GST-HHR23A~ΔUBL protein was used to saturate glutathione-sepharose resin, which was in turn used to pack an HR10/10 column. Equal amounts of HA-tagged SUMO (HA-SUMO), polyhistidine-tagged ubiquitin (Ub-His) and FLAG-tagged UBL (UBL-FLAG) were loaded simultaneously onto the column and the column was resolved in PBS. Fractions were collected and analyzed by Western blotting using epitope-specific antibodies. (B) Purified GST was bound to glutathione-sepharose resin in excess, which was then used to pack an HR10/10 column. Equal amounts of HA-SUMO, Ub-His and UBL-FLAG were mixed, loaded onto the column and analyzed as described above.

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