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. 2004 Aug;24(16):7260–7274. doi: 10.1128/MCB.24.16.7260-7274.2004

FIG. 5.

FIG. 5.

(A) PKA inhibition does not prevent ERα recruitment to the estrogen-responsive region of the CCND1 gene promoter. Sequential ChIP (ReIP) analyses of the TRE-centered region from the CCND1 promoter. Chromatin prepared from cells treated or not treated with 50 nM E2 in the absence or presence of the PKA inhibitor H89 was subjected to the ChIP procedure with an antibody against ERα (α-ER: I°IP) and then again with anti-RNA polymerase II antibodies (α-Pol II: II°IP). In some experiments, 5 μM H89 was added to the cells 30 min before E2. Input, total chromatin before the first immunoprecipitation. As a control, antibodies against total (α-CREB) or phosphorylated (αP-CREB) CREB were used to immunoprecipitate the CRE-containing proximal region of the CCND1 promoter from hormone-deprived (−E2) and hormone-stimulated (+E2) cells. (B) In vitro binding of cyclin D1 and pRb to the upstream regulatory region of the CCND1 gene. Soluble chromatin was prepared from hormone-responsive hBC cells before and at the indicated times after treatment with 50 nM E2 and immunoprecipitated with antibodies against the indicated proteins before PCR amplification. Input, total chromatin before immunoprecipitation; α-Mock, control, unrelated antibodies used for immunoprecipitation. The presence of the E2F-containing regulatory region of the c-Myc proto-oncogene was investigated with the same immunoprecipitated samples as described in Materials and Methods. (C) Sequential ChIP (ReIP) analyses of the AP/dE region from the CCND1 promoter. Chromatin prepared from cells treated or not treated with 50 nM E2 for the indicated times was subjected to the ChIP procedure with an antibody against cyclin D1 (α-D1: I°IP) and then again with anti-pRb antibodies (α-pRb: II°IP). Input, total chromatin before the first immunoprecipitation. (D) Assays like those in panel A were carried out with chromatin extracted from hBC cells stimulated at time zero with 50 nM E2 followed, where indicated (ICI), by the addition after 60 min of 5 μM ICI 182,780 to the cell culture media. The times indicated when cells were collected for analysis. Results shown are representative of multiple independent experiments. Unless otherwise indicated, data displayed are from experiments carried out with ZR-75.1 cells; identical results were obtained with MCF-7 cells.