FIGURE 3.

GM129 interacts with BMAL1 and PER2, and represses CLOCK/BMAL1-activated transcription. A, GM129 interacts with BMAL1 and PER2. Sf21 cells were co-infected with FLAG-GM129 baculovirus together with either PER2-Strep-His, BMAL1-His, or CRY1-His baculovirus. Cell extracts were prepared 48 h after infection and subjected to immunoprecipitation (IP) using FLAG resin. Immunoprecipitates were analyzed by Western blot. B, GM129 interacts with endogenous BMAL1 in an NIH3T3 cell line stably expressing FLAG-GM129. Immunoprecipitates obtained with FLAG resin from whole cell lysates were probed by immunoblotting. GM129 and BMAL1 were detected by anti-FLAG anti-BMAL1 antibody, respectively. C, HEK293 cells were transiently transfected with 25 ng of Per1 luciferase reporter construct, 2.5 ng of pBind (for normalization of transfection efficiency), and a total 250 ng of the indicated combinations of Clock, Bmal1, Gm129, and Cry1 expressing constructs together with pcDNA4. Cells were collected 48 h after transfection. Error bars represent S.D. from 3 experimental repeats. D, dose-response studies of inhibition of CLOCK·BMAL1-induced transcription by the GM129 and CRY1 proteins. Three amounts (2, 10, and 50 ng) of the Cry1 and Gm129 plasmids were used in the reporter gene assay. Experiments were done in triplicate and average values are plotted. Error bars represent standard deviation of 3 biological repeats. Note: x-axis is not set to scale.