AtCyp59 interacts with the RNA polymerase II CTD. (A) Interaction between AtCyp59 and CTD in a yeast two-hybrid assay. Indicated combinations of plasmids were cotrans-formed into yeast reporter strain, and the interaction of AtCyp59 with the CTD was assessed by growth on plates lacking histidine (–HTL) or by analysis of the activation of the second reporter gene, β-galactosidase (–HTL/β-gal). (B) In vitro interaction between AtCyp59 and CTD studied by pull-down assay. Arabidopsis CTD was overexpressed as a GST fusion in E. coli and purified (left panel). Pull-downs with AtCyp59-HA expressed in Arabidopsis protoplasts (upper right panel) or in E. coli (lower right panel). (Lanes 1) 1/10 of the input protein extracts used in pull-downs in lanes 2 and 3. (Lanes 2) pull-down with GST alone. (Lanes 3) pull-down with GST-AtCyp59. (C) In vivo interaction of AtCyp59 and CTD determined by coimmunoprecipitation. Protein extract from protoplasts transiently expressing AtCyp59-GFP were immunoprecipitated with anti-GFP antibody. Blot was probed with anti-GFP and anti-CTD (H14) antibodies simultaneously. (Lane 1) 1/ 10 of the protein extract used in IPs in lanes 2 and 3; (lane 2) immunoprecipitation with anti-GFP antibody; (lane 3) protein extract incubated with Protein A beads only.
