Figure 9

CBP enhances activation of CLOCK/BMAL1-mediated transcription by pCAF. (A) Northern blot analysis of HDAC3 mRNA expression in COS-1 cells and NIH3T3 cells. 30 μg of total RNA were loaded and blotted. (B) NIH3T3 cells were transiently transfected with pPeriod-1-Luc (10 ng), either with or without pcDNA3CLOCK (100 ng), pcDNA3BMAL1 (100 ng), pcDNA3CBP (50 ng), and pcDNA3pCAF (50 ng), as indicated. Empty vector (pcDNA3) was used to standardize the total amount of transfected DNA (410 ng). Luciferase activity was expressed as a ratio of CLOCK/BMAL1-mediated reporter activity. An asterisk indicates p < 0.05 (Student's t test). (C) COS-1 cells were transiently transfected with pHA-pCAF (2 μg), in the presence of pcDNA3CBP (6 μg). (D) COS-1 cells were transiently transfected with pmyc-CLOCK (1 μg), pmyc-BMAL1 (1 μg), or pHA-pCAF (7 μg). Empty vector (pCMV-myc or pCMV-HA) was used to standardize the total amount of transfected DNA (8 μg). (E) COS-1 cells were transiently transfected with pcDNA3CBP (6 μg), in the presence or absence of pmyc-CLOCK (0.5 μg) or pHA-pCAF (1.5 μg). Empty vector (pCMV-myc or pCMV-HA) was used to standardize the total amount of transfected DNA (8 μg). Immunoprecipitations (IP) were performed using anti-rabbit IgG, anti-CBP, or anti-HA antibodies, and then Western blot analysis was performed using anti-CBP, anti-myc, or anti-HA antibodies, as indicated. The input lanes represent 10% of total cell lysates in the binding reaction.