Figure 4. Pap1 is ubiquitylated.

To determine the ubiquitylation status of Pap1, wild type cells and as a control a pap1Δ strain were transformed to express 6His-tagged ubiquitin. The 6His-tagged ubiquitin was then precipitated using Ni²⁺ agarose under denaturing conditions in 8 M urea. The precipitates were then washed three times in a denaturing buffer and analyzed by Western blotting using antibodies to Pap1. Prior to precipitation, the protein concentrations were determined and normalized. In total 5 mg cell protein was used for each precipitation. Ubiquitylated species of Pap1 were detected in wild type cells expressing the 6His-tagged ubiquitin, but not in the pap1Δ strain or in the vector control.