Figure 5

p300 represses CLOCK/BMAL1-mediated transcription in NIH3T3 cells. (A) pVasopressin-Luc (10 ng) or (B) pPeriod1-Luc (10 ng) were transiently transfected, with or without pcDNA3CLOCK (100 ng) plus pcDNA3BMAL1 (100 ng) alone or in combination with pcDNA3p300 (100 ng), as indicated. Empty vector (pcDNA3) was used to standardize the total amount of transfected DNA (410 ng). An asterisk indicates p < 0.05 compared with lane 2 (Student's t test) (C) Cells grown in six-well plates were transiently transfected with either pSUPER or pSUPER-p300 (1.8 μg), together with either pCMV-HA or pHA-p300 1-1301 (200 ng), as indicated. HA-tagged p300 was detected by Western blotting using an anti-HA antibody. (D) Cells were transfected with either pE-less or pE-box (2 ng), together with either pSUPER or pSUPER-p300 (100 ng), and with or without pcDNA3CLOCK (100 ng) plus pcDNA3BMAL1 (100 ng) alone or in combination with pcDNA3p300 (200 ng), as indicated. Empty vector (pcDNA3) was used to standardize the total amount of transfected DNA (402 ng). An asterisk indicates p < 0.05 (Student's t test, n = 9).