ID2 restricts CLOCK and BMAL1 nuclear localization. HEK293 cells were co-transfected with either CLOCK-HA and BMAL1 (CB state), or CLOCK-HA, BMAL1, and ID2-FLAG (ICB state). A, CLOCK and BMAL1 localization (green and red, respectively) and co-staining (yellow) when overexpressed together and in the absence of overexpressed ID2. B, CLOCK and BMAL1 localization and co-staining in the presence of overexpressed ID2. Cellular protein localizations were visualized using anti-HA (for CLOCK) and anti-BMAL1 antibodies. Scale bar, 20 μm. C–E, quantification of localization data are mean ± S.E. for three independent experiments each comprising ≥100 cell counts. CB, ectopic expression of CLOCK and BMAL1 plasmids; ICB, ectopic expression of ID2, CLOCK, and BMAL1.