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. Author manuscript; available in PMC: 2013 Jun 11.
Published in final edited form as: Oncogene. 2012 Jan 9;31(43):4647–4654. doi: 10.1038/onc.2011.597

Figure 2.

Figure 2

ChIP analysis reveals restricted and cell-type-specific PU.1 and Pol II binding. A schematic of the regions examined in the ChIP assays is shown. The Conserved Elements are labeled below and approximate locations of forward primers used for QPCR analysis are labeled above. (a), Anti-PU.1 ChIP analyses are shown. ChIP assays were performed using anti-PU.1 antibody (sc-352 from Santa Cruz). ChIP assays were performed based on Upstate’s protocol exactly as described previously (14). Data shown in all ChIP panels are from three or more independent experiments. Orange and purple bars in each panel show data for positive control regions of reported PU.1 target genes Mef2c and Il7r. Panels with red bars are from myeloid cells (RAW264.7). Panels with blue bars are from pre-B-cells (NFS-25). Panels with green bars represent data from pro-T-cells (Adh.2C2). Primer pairs used for each region are labeled on the x-axis and the primer sequences are reported in (14). ChIP enriched DNA was analyzed by QPCR in triplicate for each experiment. Individual ChIP experiments were normalized first to input DNA then normalized against a region that lacked enrichment to provide relative fold enrichment. Independent experiments were then averaged. Error bars show standard deviations. Iterative one-way ANOVA was used to analyze ChIP data. ANOVA was initially performed with all regions. If ANOVA generated a p-value < 0.01, data for “regions of interest” were removed from the initial ANOVA test group and ANOVA was repeated on data from the remaining regions until these generated p>0.1. The regions remaining in the test group that lacked a statistically significant enrichment difference were then used as a control group against which to compare the removed “regions of interest” individually by T-tests. The resulting p-values were adjusted using the Bonferroni correction method and regions with p < 0.0005 were marked by asterisks in ChIP data figures. (b) Elf-1 ChIP assays using anti-Elf-1 antibody sc-631 are shown as described. (c) GABPα ChIP assays using anti-GABPα antibody sc-22810 are shown as described. (d) Ets-1 ChIP assays using anti-Ets-1 antibodies sc-22802 and sc-111are shown as described. (e) RNA polymerase II ChIP analyses are shown as described using anti-RNAPoll II antibody ab5408 from AbCam.