Fig. 3.

The CRY2-NR interaction is disrupted by mutations near the secondary pocket of CRY2. (A) CRY1/2 hybrid constructs (red: CRY1; blue: CRY2). CC, coiled coil; CT, C-terminal tail. (B and C) Co-IP of FLAG-CRY hybrids with V5-PXR (B) and V5-CAR (C) from transfected cells. (D) Repression of BMAL1:CLOCK by CRY1, CRY2, and CRY2*. U2OS cells transiently expressed Per2-luciferase, Bmal1, Clock, and Cry1, Cry2, or Cry2*. mCherry was used as a negative control. Luminescence was normalized to β-Galactosidase activity. (E) CRY2 (PDB ID code 4I6J). A, B, and C domains are light blue, dark blue, and gray, respectively. Amino acids mutated in CRY2* are red. (F) Co-IP of FLAG-CRY1, -CRY2, and -CRY2* with V5-PXR and -CAR from transfected cells. In D, data represent the mean + SD for five to six replicates per condition from one of three experiments. n.s., not significant; ***P < 0.005 vs. BMAL1:CLOCK by t test.