Figure 4

Influence of PER2 and CLOCK on CRY1/2-BMAL1 interactions. (A) and (B) HER911 cells were co-transfected with pFR-luc, pCMV-lacZ, pSCT1-Gal4-Bmal1 and 2 μg pSCT1-Per2. To obtain the indicated fold excess of pSCT1-Per2, 0.1, 0.04, 0.02, 0.01, 0.004 and 0.002 μg pSCT1-Cry1-VP16 (A) or pSCT1-Cry2-VP16 (B) were co-transfected (solid circles). Each dose of pSCT1-Cry1/2-VP16 was also transfected in the absence of pSCT1-Per2 (open circles) to assess the effect of PER2 on CRY1/2-BMAL1 interactions. For each experiment (n = 3), values obtained for cells transfected with the highest amount of pSCT1-Cry1/2-VP16 but without pSCT1-Per2 were set to 1. Note that the amounts pSCT1-Cry1/2-VP16 transfected are reversely plotted on the X axis, so that fold excess pSCT1-Per2 increases from left to right (see numbers below the graphs). (C), (D) and (F) HER911 cells were co-transfected with pSCT1-Gal4-Bmal1, pEGFP-N3 and either pSCT1-Cry1-VP16 (C and F) or pSCT1-Cry2-VP16 (D). For (C) and (D), transfections were performed with or without 2 μg pSCT1-Per2, for (F) 0.1, 0.3 or 1 μg pSCT1-Clock were co-transfected. The amount of CRY1/2-VP16, GAL4-BMAL1 and GFP was determined by Western blotting of total lysates. CRY1/2-VP16 and GAL4-BMAL1 were normalized to GFP to correct for transfection efficiency. For all proteins, values obtained for cells transfected with pSCT1-Cry1/2-VP16, pSCT1-Gal4-Bmal1 and pEGFP-N3 only were set to 1 for each experiment (n = 3). Blots are representative results from one experiment. (E) HER911 cells were co-transfected with pFR-luc, pCMV-lacZ, pSCT1-Gal4-Bmal1, pSCT1-Cry1-VP16 and the indicated amounts of pSCT1-Clock. For each experiment (n = 3), values obtained for cells transfected with pSCT1-Gal4-Bmal1 and pSCT1-Cry1-VP16 only were set to 1.