Fig 4.

Association of Sen1 with phosphorylated forms of Rpb1. Cells expressing epitope-tagged cMyc-Sen1 or cMyc-sen1-R302W were incubated with phosphospecific antibodies against Rpb1-Ser₂-P (A), Rpb1-Ser₅-P (B), or Rpb1-Ser₇-P (C) in the absence (−) or presence (+) of RNase and analyzed by Western blotting. Membranes were probed with antibodies against the cMyc epitope tag. The amount of copurified Sen1 was measured by calculating the ratio of cMyc-Sen1 or cMyc-Sen1-R302W to the Ser₂-, Ser₅- and Ser₇-phoshorylated forms of the Rpb1 CTD. In cells expressing Sen1-R302W, the addition of RNase results in a drastic decrease in the amount of Sen1 that copurifies with Rpb1-Ser₂-P (A). The sen1-R302W mutation had no effect on the amount of Sen1 copurifying with Rpb1-Ser₅-P or Rpb1-Ser₇-P regardless of RNase being present (B and C). Error bars and P values are based on three experiments.