Figure 4. RelB represses CLOCK:BMAL1 transactivation potential. (A) Effect of RelB on CLOCK:BMAL1 dependent transcription. Vectors expressing CLOCK and BMAL1 (C/B) were cotransfected with a construct containing the mPer1-luc promoter, with or without RelB. The total DNA amount was kept constant by adding carrier plasmid DNA. After normalization for transfection efficiency using β-galactosidase activity, reporter gene activity was expressed as relative luciferase units (RLU) (activity of the control transfected only with non-expressing plasmid was set to 1). All the values are the mean +/− SD (n = 3); (**) p < 0.01 (B) The E box promoter element mediates RelB repression of CLOCK:BMAL1. Experimental condition was as in A except that a reporter construct containing three copies of the Ebox consensus sequence was used (Ebox-luc). All the values are the mean +/− SD (n = 3); (***) p < 0.001. (C) Additive effect of RelB and cry1 repression on CLOCK:BMAL1 dependent transcription. Experimental conditions are as in A except that Cry1 was cotransfected with C/B or C/B + RelB. All the values are the mean +/− SD (n = 3); (**) p < 0.01, (***) p < 0.001. (D) RelB repression is CRY-independent. CLOCK and BMAL1 were cotransfected as in A in wild-type (WT) and CRY1/2 KO MEFs. % of RelB repression of CLOCK:BMAL1 transactivation is shown. All the values are the mean +/− SD (n = 3). (E) Effect of RelB and p52 on CLOCK:BMAL1 dependent transcription. Vectors expressing CLOCK and BMAL1 (C/B) were cotransfected with a construct containing the Per1-luc promoter, with or without RelB and p52, as described. After normalization for transfection efficiency using β-galactosidase activity, reporter gene activity was expressed as relative luciferase units (RLU) (activity of the control transfected only with non-expressing plasmid was set to 1). All the values are the mean +/− SD (n = 3); (**) p < 0.01, (***) p < 0.001.