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. Author manuscript; available in PMC: 2015 Jan 9.
Published in final edited form as: Mol Cell. 2014 Jan 2;53(1):148–161. doi: 10.1016/j.molcel.2013.12.003

Figure 4. Ubiquitination of Grr1 candidate substrates.

Figure 4

(A) Short and long exposures of anti-Myc Western blots of a CHX chase assay of the uncharacterized ORF YKR045C in GRR1 and grr1Δ cells. Steady state levels of Ykr045c are shown in G1- and nocodazole-arrested cells. (B) Six candidate Grr1 substrates were expressed in cells containing Ligase Traps of Grr1 as well as Mfb1 and/or Ufo1 as negative controls. Western blots of two-step purifications, as in Figure 1D, are shown. (C) Western blots of whole cell extract (I) and anti-Flag pulldowns (P) from strains expressing Myc-tagged candidate Grr1 substrates and transformed with either empty vector (pRS426) or pYES2-Grr1ΔF-Fl (a galactose-inducible copy of Grr1-Flag lacking the F-box domain).