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. Author manuscript; available in PMC: 2015 Dec 12.
Published in final edited form as: J Mol Biol. 2014 Oct 13;426(24):4049–4060. doi: 10.1016/j.jmb.2014.10.004

Fig. 4.

Fig. 4

Stabilization of proteolytic substrates by Rad23 phospho-mimetic mutants. (a) overexpression of the Rad23 UbL domain can interfere with protein degradation. Ub-Pro-β-gal was co-expressed with either GST-UbL or GST-UbLS73D. Ub-Pro-β-gal was detected in total extract during a cycloheximide chase, by immunoblotting, and moderate stabilization was observed. (b) Ub-Pro-β-gal was co-expressed with GST-UbL or the double mutant GST-UbLS47ES73D in wildtype yeast Pab1 levels were determined as a loading control. These results are representative of three independent experiments. (c) The reporter substrate Ub-Pro-β-gal was co-expressed with Flag-Rad23, Flag-Rad23S47AS73A, or Flag-Rad23 S47ES73D in rad23Δ. β-galactosidase activity was measured in whole cell extracts to estimate Ub-Pro-β-gal stability. β-gal activity representing the average of four independent experiments were normalized to the value detected in the wildtype strain.