Fig. 6. Recruitment of Pbx1 and Hox11 to the mouse Hox11 promoter. (A) Schematic illustration of 1.2 kb of the Hox11 genomic segment with known promoter activity that contains Pbx1-binding sites, and a 5' upstream region. Primers used for PCR analysis are indicated by arrows (each pair in a different color) and the oligoprobe (PX1) used for EMSA is indicated by a black box. (B) Binding of Pbx1 and Hox11 to the (PX1) oligo within the Hox11 promoter. Nuclear extracts derived from wild-type primary embryonic spleen cells were subjected to EMSA with a radiolabeled PX1 probe containing a Pbx1 wild-type (PX1) or mutated (mPX1) core binding site (underlined), as indicated above gel lanes. Asterisk indicates non-specific band. (C) Primary cells isolated from embryonic spleen stained in culture for the mesodermal marker smooth muscle actin (green fluorescence). Western blot analysis (panel below) demonstrates that these cells produce Pbx1, Prep1 and Hox11 proteins. Two isoforms of Hox11 are present in embryonic spleen, as indicated (Yamamoto et al., 1995). (D,E) For ChIP analysis, chromatin was subjected to immunoprecipitation (IP) using antibodies specific for Pbx1b (anti-Pbx1b) (D) or Hox11 (anti-Hox11) (E). As negative controls, IPs were also performed with an anti-GFP antibody, rabbit serum (RS) or no antibody (No Ab). A primer pair that amplifies a region within the Bmp4 promoter was used as an additional negative control. (F) Synergistic activation of the Hox11 promoter by Pbx1, Prep1 and Hox11 proteins. Luciferase activity was assayed from transiently transfected NIH 3T3 cells. Co-transfection assays were performed in the presence (+) of the indicated expression vectors encoding Pbx1, Prep1 or Hox11, and with a vector containing the promoter regulatory region of Hox11 (p540), or with vector alone (pGL2). Data are expressed as the fold activation over the p540 basal luciferase activity. Bars represent the mean of three independent transfections (performed in duplicate)±s.e.m. normalized for ß-galactosidase activity (internal control) within each experiment.