Inhibition of CCR5 transcription and CCR5 promoter activation by forskolin and ICER. (A) mRNA expression levels of CCR5, ICER, and CREB in T lymphocytes, DCs, and microglia treated with forskolin (Forsk; 20 μM) for 6 hours, as determined by RT-PCR. Forskolin treatment reduces amounts of CCR5 transcript in T lymphocytes. Shown are representatives of at least 2 independent experiments. (B) Transient transfection of the CREB-responsive CCR5 downstream promoter construct CCR5-pB3 with CREB-1 and ICER expression vectors, showing inhibition of CREB-1-induced CCR5 promoter activity by ICER. Depicted are relative light units (RLU) per second obtained for luciferase activity and normalized with Renilla luciferase activity expressed as means plus or minus SEM. Shown is a representative of 3 independent experiments performed in triplicate. (C) Chromatin immunoprecipitation (ChIP) analysis of CREB-1, NF-κB, and IRF-1 binding, and acetylated histone H3 (AcH3) and RNA polymerase II (RNApolII) binding to the CCR5 and GAPDH promoter regions in T lymphocytes treated with forskolin (Forsk; 20 μM) for 6 hours. Protein binding is depicted as percentage enrichment, relative to input chromatin and corrected for background binding. Shown are means plus or minus SEM of 2 PCR reactions of a representative of 3 independent experiments.