Fig. 1. Ldb1 and Ssdp2 specifically interact in vitro. Ldb1 (from mouse), Ssdp2 (human) and the LIM-HD protein Lhx3 (mouse) were transcribed and translated in rabbit reticulocyte lysates. Proteins labeled with ³⁵S-methionine and/or tagged with the FLAG epitope were mixed in the combinations shown above each lane and then complexes were immunoprecipitated with anti-FLAG antibody-conjugated agarose beads (see Materials and Methods). Ssdp2 protein is efficiently immunoprecipitated by Ldb1 (lane 2), but not by Lhx3 (lane 4). Lhx3 can bind Ldb1 (lane 1) and can immunoprecipitate Ssdp2 in the presence of Ldb1 (lane 3). This indicates the formation of a ternary complex in which Lhx3 and Ssdp2 are each bound to Ldb1, but they do not directly interact with one another. Control experiments show that the Ldb1-Ssdp2 complex is not immunoprecipitated in the absence of the FLAG epitope (lane 5), and only a small amount of either Ssdp2 or Ldb1 binds non-specifically to the beads (lanes 5 and 6).