The HBA genes are repressed by histone deacetylases in nonerythroid cells. (A) Semiquantitative RT-PCR analysis of HBA2, ACTB, and LUC7L cDNA in the cell lines indicated that were either mock-treated, or treated with 0.2 μmol/L TSA for 24 hours. (B) Northern blot analysis of RNA from the cell lines indicated that were either mock-treated (−) or treated with 0.2 μmol/L TSA for 24 hours (+). The membrane was hybridized first to a probe which recognizes transcripts from both HBA1 and HBA2 genes (top panel) and subsequently to an ACTB cDNA probe as loading control (bottom panel). (C) Real-time RT-PCR analysis of HBA2 and HBB cDNA in EBV-lymphocytes that were either mock-treated or treated with 0.2 μmol/L TSA for 4 or 8 hours. Results are shown as fold change relative to mock-treated cells, after normalizing for GAPDH signal. (D) Real time RT-PCR analysis of HBA2 cDNA in EBV-lymphocyte cultures that were either mock-treated or treated with 0.2 μmol/L TSA for 8 hours, in the presence of either no secondary drug or actinomycin D or cycloheximide as indicated. Results are shown as fold change relative to mock-treated cells (with no secondary drug), after normalizing for GAPDH signal. Shown is the mean of replicate experiments, with error bars indicating the range. For each experiment, PCR was performed in triplicate. (E) ChIP experiments were carried out on primary T lymphocytes or proerythroblasts using an antibody against HDAC1, and material was analyzed at the amplicons indicated surrounding the α-globin locus (shown in Figure 1) or a control amplicon within the 18S rRNA gene. Shown are the mean values obtained from replicate experiments with error bars indicating the range.