Figure 5.

Interaction of Mad and Dad with Tkv. (A) Affinity cross-linking using ¹²⁵I-BMP-2 was performed to examine the interaction of Mad and Dad with Tkv. Cells were transfected with FLAG-tagged Mad (lanes 1 and 2) or Dad (lanes 3 and 4) with combinations of wild-type (WT; lanes 1 and 3) or kinase-defective (KR; lanes 2 and 4) Tkv and Punt and affinity labeled with ¹²⁵I-BMP-2. The lysates were subjected to immunoprecipitation with anti-FLAG antibody and detection by SDS-PAGE. Expression levels of proteins were monitored by Western blotting. (B) Interaction of FLAG-tagged Dad with HA-tagged Tkv was examined by immunoprecipitation with anti-FLAG antibody followed by Western blotting with anti-HA antibody. Wild-type (WT), the constitutively active form (QD), and the kinase-defective form (KR) of Tkv were used. Expression levels of proteins were monitored by Western blotting. (C) Effect of Dad expression on the interaction of Mad with Tkv was examined by affinity cross-linking followed by immunoprecipitation. Mad, Dad, and receptors were tagged with FLAG, myc, and HA, respectively. For coimmunoprecipitation, anti-FLAG antibody was used. Expression levels of proteins were monitored by Western blotting.