Journal: Nucleic Acids Research

Article Title: Identification and characterization of DNA sequences that prevent glucocorticoid receptor binding to nearby response elements

doi: 10.1093/nar/gkw203

Figure Lengend Snippet: Characterization of the NRS sequences. ( A ) EMSA comparing binding of the DNA binding domain of GR to a GBS sequence flanked by either the control sequence (left) or by NRS2 (right). ( B ) DNase I accessibility assay was performed with populations of transgenic cells with stably integrated reporters as indicated. Regions of interest were analyzed by qPCR ( FKBP5 : control accessible region; IGFBP1 : control inaccessible region; integr. GBS: integrated reporter region). Results are shown as % of input remaining after DNAse I digestion ±SEM (n = 3). ( C ) Nucleosome occupancy was analyzed using micrococcal nuclease (MNase) assays for populations of transgenic cells with stably integrated reporters as indicated. Regions of interest were analyzed by qPCR. Integr. GBS: integrated reporter region. Results are shown as % of input remaining after MNase digestion ±SEM (n = 3).

Article Snippet: For each sample containing 0.8 μg genomic DNA in 50 μl storage buffer, an equal volume of MNase reaction buffer (50 mM Tris pH 7.4; 25 mM KCl; 2.5 mM CaCl2 ; 5 mM MgCl2 ; 12.5 % glycerol) was added containing 1 μl MNase (NEB; ∼200 kunitz) for MNase-treated samples.

Techniques: Binding Assay, Sequencing, Transgenic Assay, Stable Transfection, Real-time Polymerase Chain Reaction