Interaction of AtCyp59 with SR proteins. (A) Coomassie blue–stained gel of purified recombinant GST-AtCyp59. Molecular mass standards in kilodaltons are indicated on the left. (B) AtCyp59 interacts with SR proteins in vitro. Whole cell extracts from protoplasts transiently expressing HA-tagged SR proteins were incubated with glutathione Sepharose beads coated with GST-AtCyp59. After washing, proteins retained on the beads were analyzed by SDS-PAGE and Western blotting with rat anti-HA monoclonal antibody. (Lanes 1) 1/10 of the input extract used for pull-downs in lanes 2 and 3; (lanes 2) pull-downs with beads coated with GST alone; (lanes 3) pull-downs with beads coated GST-AtCyp59. (C) Schematic representation of AtCyp59 deletion mutants fused to GST. (D) Coomassie blue–stained gel of purified recombinant GST-AtCyp59 deletion mutants. Molecular mass standards in kilodaltons are indicated on the left. (E) Interaction of AtCyp59 deletion mutants with SR proteins. Pull-down experiment performed with GST-AtCyp59 deletion mutants and one member of each Arabidopsis SR protein subfamilies. (Lane 1) 1/10 of the input extract used for pull-downs in lanes 2–6; (lane 2) pull-downs with beads alone; (lane 3) pull-downs with beads coated GST-AtCyp59; (lane 4) pull-downs with beads coated GST-AtCyp59D1; (lane 5) pull-downs with beads coated GST-AtCyp59D2; (lane 6) pull-downs with beads coated GST-AtCyp59D3.
