Fig 3. Guk1-7 degradation is proteasome dependent.

(A) Flow cytometry profiles of wild type cells expressing Guk1-GFP or Guk1-7-GFP were incubated at 25°C or 37°C for two hours in the presence of CHX. Fluorescence in cells with the control empty vector (EV) are also shown. Lines demark median GFP fluorescence values and i denotes the difference in median intensity values used to measure protein stability. (B) Comparison of quantitation of Guk1-7 levels in a CHX chase assay by Western blot or flow cytometry. (C) Wild type and rpt6-20 cells expressing Guk1-7-GFP were incubated with CHX at 25°C or 37°C and samples were analysed by flow cytometry at the indicated time points. The results represent the means and standard deviations of three independent experiments. (D) Guk1-7-GFP expressing wild type or pep4∆prb1∆ cells were incubated at 25°C or 37°C in the presence of CHX and samples were analyzed by flow cytometry at the indicated time points. The results represent the means and standard deviations of three independent experiments. (E) rpt6-20 cell expressing Guk1-GFP or Guk1-7-GFP were grown at 25°C and then shifted to 37°C for 1 hour prior to their fixation and imaging. Scale bar represents 5μm. (F) Guk1-GFP and Guk1-7-GFP expressing cells were incubated at 25°C and cell lysates were immunoprecipitated using GFP-Trap beads and then immunoblotted with anti-ubiquitin, anti-GFP, and anti-Pgk1 antibodies.