Journal: PLoS ONE

Article Title: Identification of She3 as an SCF Grr1 Substrate in Budding Yeast

doi: 10.1371/journal.pone.0048020

Figure Lengend Snippet: (A) Structure of Grr1 and the deletion mutants used in this study. (B) Interactions of Grr1 mutants with several proteins that appear not to be SCF Grr1 substrates. (C) Degradation of Prp3, Yir016w, Rri2 and Fob1 in the indicated strains. WT: YJB15; grr1Δ : DOY805. Proteins with the indicated tags were expressed from a Gal promoter for 50 min. Samples were collected at the indicated times after adding cycloheximide and glucose to the cultures and analyzed by immunoblotting against the epitope tag. (* indicates a nonspecific band recognized by the anti-Myc antibody.) Proteins were expressed from the following plasmids (Supplemental Table 1): pRW0511083 (Prp3), pRW0511085 (Yir016w), pRW1022081 (Rri2), and pRW1103083 (Fob1). (D) Interaction patterns of Grr1 substrates (Pfk27 and Gic2) with the Grr1 mutants. (E) Interaction pattern of She3 with the Grr1 mutants. (F) The degradation of She3 was analyzed in wild-type cells (YJB15), grr1Δ cells (DOY805), and grr1Δ cln1Δ cln2Δ cells (DOY855). Cdc28 was used as a loading control in (C) and (F).

Article Snippet: 2% galactose was added and cultures were grown for 50 min to induce protein expression, followed by addition of 2% dextrose and 500 μg/ml of cycloheximide (Acros Organics).

Techniques: Western Blot, Control