<i>MIP/AQP0</i> region demonstrates enrichment in chromatin immunoprecipitation experiments with PITX3 or FLAG antibody.
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Abstract
<p><b>A.</b> Endogenous PITX3 is bound to the proximal <i>MIP/AQP0</i> promoter in human lens epithelial (HLE) cell cultures. Chromatin immunoprecipitation assays were performed using untransfected HLE cells and human PITX3 or IgG (control) antibodies. The samples were analyzed by semi-quantitative PCR using <i>MIP/AQP0</i> proximal promoter- specific primers and negative control primers. Please note robust amplification of <i>MIP/AQP0</i> promoter region from ChIP sample precipitated with PITX3 but not with control IgG antibody (red arrow) and equal levels of DNA amplification for negative control region in both samples (black arrowhead). <b>B.</b> PITX3_FLAG is bound to proximal <i>MIP/AQP0</i> promoter in HLE cell cultures following transfection with PITX3-FLAG expression plasmid. HLE cells were transfected with either PITX3-FLAG expression plasmid or control pcDNA3.1 expression vector. ChIP assays were performed with FLAG-M2 or control IgG antibody. The ChIP samples were analyzed by semi-quantitative PCR as described in A. Please note enrichment of <i>MIP/AQP0</i> promoter region in ChIP sample obtained from PITX3-FLAG transfected cells and precipitated with FLAG antibody in comparison to FLAG-precipitated ChIP sample obtained from pcDNA3.1 transfected cells as well as IgG-precipitated ChIP sample obtained using either PITX3-FLAG or pcDNA3.1 transfected cells (red arrow). In addition to this, amplification of negative control region demonstrated similar levels across all samples (black arrowhead). <b>C</b> and <b>D.</b> Statistical analysis of multiple semi-quantitative PCR/ChIP experiments performed in untransfected (C) and transfected HLE cells (D) as described in A and B, correspondingly. Presence of <i>MIP/AQP0</i> promoter or negative control region DNA in various ChIP samples was evaluated by semi-quantitative PCR followed by densitometric analysis and expressed as a percentage of input values; mean and standard deviation for at least 3 independent experiments were calculated and analyzed by Student's t test. Please note statistically significant enrichment for <i>MIP/AQP0</i> promoter region DNA precipitated with PITX3 (C) or FLAG (D) antibody in comparison to control IgG-precipitated chromatin in HLE untransfected (C) or transfected (D) cells. IgG = normal mouse IgG; PITX3 = PITX3 polyclonal antibody; FLAG = anti-FLAG monoclonal antibody.</p