Figure 1

CBP represses CLOCK/BMAL1-mediated transcription in NIH3T3 cells. (A) Cells were transiently transfected with either pE-box (black-bar) or pE-less (white-bar) (2 ng), either with or without pcDNA3CLOCK (100 ng) and pcDNA3BMAL1 (100 ng), in the presence of increasing amounts (0, 5, 10, 20, 50, 100 or 200 ng) of pcDNA3CBP, as indicated. Empty vector (pcDNA3) was used to standardize the total amount of transfected DNA (502 ng). An asterisk indicates p < 0.05 compared with black-bar in lane 2 (pE-box; Student's t test). (B) Cells grown in six-well plates were transiently transfected with either pCMV-myc (1000 ng) or pmyc-CLOCK (500 ng) and pmyc-BMAL1 (500 ng), either alone or in combination with pcDNA3CBP (500 ng). Empty vector (pCMV-myc and pcDNA3) was used to standardize the total amount of transfected DNA (2 μg). 48 hrs later, transfected cells were lysed and cell lysates were subjected to Western blot analysis using anti-myc or anti-CBP antibody. Relative proteins level was presented. An asterisk indicates a P value of < 0.05 (Student's t test, n = 3). (C) pVasopressin-Luc or (D) pPeriod1-Luc (10 ng) were transiently cotransfected, with or without pcDNA3CLOCK (100 ng) plus pcDNA3BMAL1 (100 ng), in combination with increasing amounts (0, 20, 50, 100 or 200 ng) of pcDNA3CBP, as indicated. Empty vector (pcDNA3) was used to standardize the total amount of transfected DNA (510 ng). An asterisk indicates p < 0.05 compared with lane 2 (Student's t test).