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. 2009 Jan 23;4(1):e4259. doi: 10.1371/journal.pone.0004259

Figure 6. UBP2 is not necessary for cycloheximide (CHX) triggered internalization of Fur4.

Figure 6

(A) pFUR4-GFP bearing cells were grown in raffinose overnight. Galactose was added for 2 hours to induce synthesis, and glucose was then added for 10 min to chase Fur4-GFP to the plasma membrane. CHX (0.1mg/ml) was added and GFP signal examined by fluorescence microscopy and Nomarski optics. Time refers to the time after addition of CHX, with 0 min as pre-induction. (B) Uracil uptake was measured at different times after the addition of CHX in WT and ubp2Δ strains. Results are expressed as a percentage of the initial uracil uptake, and plotted on a log scale. (C) Fur4 ubiquitin profile at the plasma membrane is unchanged in ubp2Δ mutants. pFUR4 was transformed into WT, ubp2Δ, rup1Δ, and rsp5-1, and cells, which were grown in raffinose overnight. Expression from pFUR4 was induced for 90 min with galactose before adding glucose for 15min to chase Fur4 to the plasma membrane. Total protein extracts (lysate) and enriched membrane fractions were collected and analyzed by Western blotting to visualize Fur4 and Fur4-ubiquitin conjugates. 3-phosphoglycerate kinase (PGK) and porin, a mitochondrial membrane protein, were used as loading controls.