Fig. 2
From: Whi7 is an unstable cell-cycle repressor of the Start transcriptional program
Analysis of Whi7 degradation in ubiquitin-ligase mutant strains. a–e Cells of the pre1 pre2 (JCY1740) and its parental WCG4α (JCY1739), cdc53 (JCY1732), grr1 (JCY1760), cdc4 (JCY1757) and their parental W303 (JCY1728), and cdc16 (JCY1737) and its parental A364 (JCY1735) strains expressing a HA-epitope-tagged Whi7 protein at endogenous level were transferred at 37 °C for 3 h (except the grr1 mutant and its parental strain) and then incubated in the presence of 100 μg mL−1 cycloheximide (chx). Whi7 protein level was analyzed at the indicated time after the addition of cycloheximide by western blot. Cdc28 is shown as loading control. Graph represents the relative amount of Whi7 protein related to Cdc28. f The grr1 mutant strain (JCY1760) was co-transformed with a plasmid expressing a flag-tagged Grr1 lacking its F-box (Grr1ΔF) and either the pWHI7 or pWHI7-NP plasmid (expressing HA-tagged wild-type or a non-phosphorylatable Whi7 respectively) or a control vector. Cells were grown on raffinose medium and transferred to galactose medium for 4 h. Whi7 was immunoprecipitated (IP) from crude extracts and the presence of Grr1ΔF and Whi7 in the input, unbound and immunoprecipitated fractions was determined by western analysis. A longer exposition of the vector samples is shown for better evaluate background signal
