Figure 3.
A direct interaction between Gro and Rpd3. (A) Copurification of baculovirus-expressed Flag-tagged Gro (M2Gro) and six-histidine-tagged Rpd3 (H6Rpd3). Nuclear extracts (NE) prepared from insect cells expressing either M2Gro, H6Rpd3, or both together were incubated with Ni2+–NTA–agarose (Ni) or anti-Flag affinity beads (M2). After extensive washing of beads, bound proteins were eluted with SDS, resolved by SDS-PAGE, and immunoblotted with anti-Gro (top) or anti-Rpd3 (bottom) antibodies. (B) A Coomassie blue stained SDS–polyacrylamide gel showing baculovirus-expressed and affinity-purified Flag-tagged Gro (M2Gro) and Flag-tagged Rpd3 (M2Rpd3). (C) Equal amounts of anti-Flag M2 beads (M2) or M2 beads containing purified Flag-tagged Gro (M2Gro) were incubated with 35S-labeled Rpd3 produced by in vitro translation. After subjecting the beads to extensive washing, bound 35S-labeled Rpd3 was eluted, and analyzed by SDS-PAGE and autoradiography. (D) In a reciprocal experiment, M2 beads containing purified M2Rpd3 were used to examine the interaction with 35S-labeled Gro. (1/10 IP) Ten percent of total 35S-labeled protein input.