DREAM interacts with the Ct region of TSHR. A, Coimmunoprecipitation of DREAM and TSHR with a monoclonal antibody for DREAM in whole-cell extracts of thyroid glands from wild-type and transgenic mice (LeuBTA15). A nonrelated antibody (anti-HA) was used as a negative control. TSHR and DREAM were detected by Western blot. Note that transgenic mice contain higher levels of TSHR and total DREAM proteins, as expected. Equal amounts of total protein extracts were used in both cases. B, Coimmunoprecipitation of DREAM-HA and TSHR with an anti-HA antibody in whole-cell extracts from TSHR-10,000 cells transfected with the indicated plasmids. TSHR and DREAM were detected by Western blot. White arrowhead indicates Ig chain. C, Schematic representation of the TSHR protein and deletion fragments containing the indicated Nt, intracellular (TM/Ct), and Ct regions tagged with Myc. Extracellular domains (gray), transmembrane domains (black), and intracellular domains (white) are indicated. D, Coimmunoprecipitation of DREAM-HA and the Myc-tagged TSHR deletion fragments with an anti-HA antibody in whole-cell extracts from CHO-K1 cells transfected with the indicated plasmids. Western blots were developed with anti-Myc and with anti-DREAM as a control. Input corresponds to 5% of total protein. White arrowhead indicates Ig chain.