FIGURE 2.

Npa3 depletion affects transcription in vivo, but not in vitro. A, depletion of Npa3-degron after inducing degradation by raising the temperature to 37 °C. No degradation of Npa3 is observed in a WT congenic strain (not shown). B, Rpb1 ChIP using WT or Npa3-degron cells at the non-depleting temperature (25 °C) or depletion-inducing temperature (37 °C). ChIP signals on ZPS1 and NAT4 are expressed as the percentage of input. Error bars denote S.D. of three biological replicates. C, Western blots of extracts from an Npa3-FLAG-TEV₂-Myc₁₈ strain before (titration on left) or after depletion of Npa3 by 9E10 (anti-Myc) or control beads (Mock). Note that the Npa3 signal after 9E10 depletion (lane 7) is similar to 1–5% of the signal before depletion (lanes 4 and 5), whereas the signal in the mock depletion is similar to 100% (compare lane 6 with lane 1). D, promoter-driven transcription in mock- or Npa3-depleted extract in the absence or presence of added recombinant Npa3.