Figure 1.

CREB inhibits GRIP1-stimulated activation of the GAL4-reporter gene and reduces GRIP1 protein level. A, COS-1 cells were transiently transfected with a GAL4-responsive luciferase reporter construct (GAL4-luc) (1.1 μg) together with expression plasmids encoding GAL4-GRIP1 (1.0 μg) and CREB (0.01, 0.02, 0.05, 0.1, 0.25, 0.5, 1.0, and 2.0 μg) for 48 hours. Luciferase activities were measured as described in Materials and Methods. The figures show the mean ± SD of triplicate transfections from at least 3 independent experiments. B, COS-1 cells were transfected with expression plasmids encoding HA-GRIP1 (2.0 μg) and CREB (0.5, 1.0, 1.5, and 2.0 μg). Forty-eight hours after transfection, cell were lysed and analyzed by Western blotting using anti-HA (GRIP1), anti-CBP, anti-CREB, and anti-GAPDH antibodies. The results presented are representative of at least 3 independent experiments. C, COS-1 cells seeded in phenol red-free DMEM containing charcoal-stripped FBS were transfected with expression plasmids encoding human ERα (0.1 μg), HA-GRIP1 (1.0 μg), and CREB (0.1 μg) together with the ERE-TATA-luc reporter construct (1.1 μg). The cells were treated with vehicle or 17β-estradiol (E2, 0.1μM) for 24 hours. Luciferase activities were measured 48 hours after transfection. The figure shows the mean ± SD of triplicate transfections (**, P < .01, Student's t test) and is representative of 3 independent experiments.