Fig. 6. Mouse Cited2 and Wt1 interact physically. (A) Wt1 and Cited2 endogenous proteins interact physically in M15 cells. Nuclear proteins from M15 cells were immunoprecipitated with antibodies for Wt1 (IP Wt1) or ß-galactosidase (IP ßGal, specificity control). Cited2 (C) was only detected in the immunoprecipitate formed with Wt1 antibody. `5%' means 5% of the amount of protein used for immunoprecipitation. (B) Wt1 and Cited2 interact physically in transfected cells. 293A cells were transfected with expression vectors for Wt1(-/-) or Wt1(+/+) and Cited2. Proteins were immunoprecipitated with antibodies raised against Wt1 (left panel) or Cited2 (right panel). Non-immune rabbit IgGs or rabbit anti-Sf-1 antibody did not immunoprecipitate Cited2 in control experiments. EV, empty vector; C, western blot with Cited2 antibody; W, western blot with Wt1 antibody. (C) Cited2 interacts directly with the Wt1 DNA-binding domain. In vitro translated ³⁵S-labelled Cited2 protein (IVT Cited) was incubated with GST alone (GST, negative control) or immobilised GST-tagged full-length (FL), N-terminal (N-ter) or zinc-finger DNA-binding domain (ZF) fragments of Wt1(-/-) protein. After washes, bound proteins were eluted and separated on a SDS-PAGE gel and detected by autoradiography (upper panel). A Coomassie staining of the gel is provided as a loading control for the amounts of the GST fusion proteins (lower panel). Arrowheads indicate the GST-fused Wt1 proteins.