Figure 1
Magel2 represses Clock:Bmal1-mediated transcription at an E-box promoter. Dual luciferase transcription assays following transfection of NIH3T3 cells with the mPer2:luc reporter plasmid and a renilla internal control plasmid, together with combinations of expression plasmids variously encoding epitope-tagged Clock, Bmal1, Magel2, Cry1, and Per2 proteins. Data are normalized to the baseline activity of the normalized mPer2:luc reporter. A * indicates a significant difference from the baseline mPer2:luc activity, and a # indicates a significant repression compared to Bmal1:Clock-mediated activation (p < 0.05). These results represent one of three experiments completed in triplicate. Error bars represent the standard error of the mean.
Figure 2
Magel2 interacts with Bmal1 and Per2. A) HEK293 cells were transfected with expression plasmids encoding HA-Bmal1, Xpress-Magel2, or both constructs. Five percent of the volume of cell lysate used for immunoprecipitation was immunoblotted to confirm the presence of HA-Bmal1 and Xpress-Magel2 protein in the appropriate input samples (top). The protein complexes were immunoprecipitated with anti-HA or anti-Xpress antibodies, and the immunoprecipitates detected by immunoblotting with antibodies directed against the reciprocal epitope tag (bottom). B) HEK293 cells were transfected with expression plasmids encoding V5-Per2, Xpress-Magel2, or both constructs. Five percent of the volume of cell lysate used for immunoprecipitation was immunoblotted to confirm the presence of Xpress-Magel2 and V5-Per2 protein in the appropriate input samples (top). The protein complexes were immunoprecipitated with anti-V5 or anti-Xpress antibodies, and the immunoprecipitates detected by immunoblotting with antibodies directed against the reciprocal epitope tag (bottom).
Figure 3
Subcellular localization of circadian proteins in the presence of Magel2. pXpressMagel2 and plasmids encoding other epitope-tagged circadian proteins (A, Clock, B, Bmal1, C, Per2, and D, Cry1) were detected in co-transfected NIH3T3 cells by immunofluorescence. Plots of fluorescence intensity were generated to assess overlap between the immunofluorescence signals from each protein within transfected cells, which were also stained with Hoechst 33342 to label the nucleus.
Figure 4
Subcellular distribution of circadian proteins is modified by co-expression of Magel2. NIH3T3 cells were transfected with combinations of Xpress-Magel2 and circadian expression constructs. Transfected cells were scored as having the majority of the fluorescent signal either in the nucleus or in the cytoplasm. Differences in the nuclear/cytoplasmic distribution between transfections were detected using a Fisher exact test, with p < 0.05 deemed statistically significant. A) Example of primarily nuclear (N) or primarily cytoplasmic (C) localization of epitope tagged Bmal1 (in green, nucleus is in blue). B) Localization of Clock when expressed alone (C), expressed with Bmal1 (CB), expressed with Magel2 (CM) or expressed with Clock and Magel2 (CBM). C) Localization of Bmal1 when expressed alone (B), expressed with Clock (BC), expressed with Magel2 (BM) or expressed with Clock and Magel2 (BCM).