Ldb1 knockdown inhibits enrichment of the Ldb1 complex and Ser2P RNA pol II at the β-globin promoter and LCR in MEL cells. (A) MEL cells were stably transduced with Ldb1 shRNA or with an empty virus (Ctrl) and individual clones were isolated. Reduced Ldb1 expression in 2 stable Ldb1 knockdown clones (KD1 and KD2) was confirmed by Western blot analysis before (−) and after 4 days of 2% DMSO treatment (+). GATA-1 and α-tubulin served as positive and internal controls, respectively. (B) Stable clones were treated with 2% DMSO. Total RNA was isolated at day 4, and β-globin expression was analyzed by quantitative real-time RT-PCR. Each value was normalized with 18S ribosomal RNA. Three RNA preparations were analyzed. Error bars represent the SEM. (C) ChIP was carried out using KD1 cells after 4 days of 2% DMSO induction using antibodies to acH3 and H3K4me3, and the data were graphed together by setting the highest level for each antibody equal to 1. (D) The ChIP assay was performed with KD1 cells after 4 days of 2% DMSO induction using antibodies to Ldb1, GATA-1, LMO2, and SCL. Error bars represent the SEM for multiple independent chromatin preparations. (E-F) ChIP was carried out as for panel D using antibodies to Ser2P pol II (E) or Cdk9 (F). Necdin served as a negative control. Error bars represent the SEM for multiple independent chromatin preparations. ChIP assays with KD2 cells gave similar results (not shown) to those in panels C through F.