CRY1 and CRY2 have different effects on PER2-BMAL1 interaction. (A) HER911 cells were co-transfected with pFR-luc, pCMV-lacZ, pSCT1-Per2-VP16, pSCT1-Gal4-Bmal1 and the indicated doses of pSTC-TK-Cry1/2 with or without addition of 0.3 μg pSCT1-Clock. For each experiment (n = 3), values obtained for cells transfected with pSCT1-Clock but without pSTC-TK-Cry were set to 1. (B), (C) and (D) HER911 cells were co-transfected with pSCT1-Per2-VP16, pSCT1-Gal4-Bmal1, pEGFP-N3 and 0.001, 0.01, 0.1 or 1 μg pSTC-TK-Cry1/2 (B) or 0.1 μg pSTC-TK-Cry1/2 with and without addition of 0.3 μg pSCT1-Clock (C, D). The amount of PER2-VP16, GAL4-BMAL1 and GFP was determined by Western blotting of total lysates (B, C) or nuclear and cytoplasmic fractions (D). Antibodies against CREB and HSP90 were used to verify correct cell fractionation. PER2-VP16 and GAL4-BMAL1 were normalized to GFP to correct for transfection efficiency. For both proteins, values obtained for cells transfected with pSCT1-Per2-VP16, pSCT1-Gal4-Bmal1 and pEGFP-N3 only were set to 1 for each experiment (n = 4 for B and D, n = 5 for C). Blots are representative results from one experiment.
Langmesser et al. BMC Molecular Biology 2008 9:41 doi:10.1186/1471-2199-9-41