Figure 6.
Analysis of ubiquitin synthesis rates in doa4Δ cells. (A) Northern analysis of UBI4. Total RNA was prepared from wild-type (MHY501) and doa4Δ (MHY623) cells grown in minimal media in early logarithmic phase (t = 0 h) and stationary phase (t = 8 and 14 h). UBI4 transcripts were detected using a 1.3-kb BstXI-BclI fragment from UBI4. The blot was stripped and reprobed with a 560-bp ClaI fragment from the ACT1 gene. (B) Pulse labeling of ubiquitin. Radiolabeled ubiquitin was immunoprecipitated from wild-type (MHY501) and doa4Δ (MHY623) cells in late logarithmic phase (OD600 ∼ 1.5) with anti-ubiquitin antiserum, which does not efficiently immunoprecipitate ubiquitin–protein conjugates. Cells were pulse-labeled with 35S-TransLabel for 30 min.