Figure 2

The CC-3 and MPM-2 epitopes of recombinant Rpb1 are generated by different kinases. (A) Schematic representation of the N- and C- moieties of the CTD of Rpb1 fused to GST tags for in vitro phosphorylation experiments. The N-CTD comprised the first 26 repeats of the consensus YSPTSPS and the C-CTD comprised the more degenerated last 26 repeats. (B) N-CTD and C-CTD recombinant proteins were phosphorylated with a mitotic extract or with Cdk9/cyclin T1 and subjected to SDS-PAGE and immunoblotting with CC-3 and MPM-2. Arrows indicate the position of unphosphorylated N-CTD and C-CTD controls, revealed by Ponceau red staining prior to immunostaining. Mitotic kinases strongly enhanced CC-3-reactivity in the C-CTD fragment whereas Cdk9/cyclin T1 generated more efficiently the MPM-2 epitope, especially in the N-CTD fusion protein. N: N-CTD, C: C-CTD.