FIG. 6.

Pan1 interaction is specific to the Pitx family. In experiments performed as described in the legend to Fig. 5, Pitx2 (A) and Pitx3 (B) show the same synergism as Pitx1 (Fig. 5A) but not goosecoid (Gsc, C) or Otx1 (D). Results are the average (± SEM) of at least two experiments performed in duplicate. (E) Gsc and Nkx2.5 homeoproteins do not interact with Pan1 in vitro. Pull-down assays were performed with the small in vitro-synthesized bHLH domain of Pan1 and purified MBP-Nkx2.5, MBP-Gsc, MBP-LacZ, and MBP-Pitx1. Input was 10% of the amount used. (F) An anti-Pitx1 antibody coimmunoprecipitates Pan1. Nuclear extracts were prepared from COS-1 cells transfected with Pitx1- and Pan1-expressing plasmids, and 10 μg of extracts was used for coimmunoprecipitation (CO-IP), input, and control samples. (G) Coimmunoprecipitation of Pan1 with Pitx1 from nuclear extracts of untreated AtT-20 cells. The experiment was performed as in panel F except that 200 μg of extracts was used for each sample.