Journal: Scientific Reports
Article Title: GC-elements controlling HRAS transcription form i-motif structures unfolded by heterogeneous ribonucleoprotein particle A1
Figure Lengend Snippet: ( A ) ChIP experiment to determine the occupancy of hras -1, hras -2 and control sequence (870 bp downstream from TSS) by hnRNP A1. Histograms shows the relative occupancy of hras -1 and hras -2 by hnRNP A1, RNA Pol II (positive control) and IgG (negative control). Data have been normalized by IgG signal; ( B ) EMSA of 32 P-labelled hras -1 Y and hras -2 Y in 50 mM Tris-acetate pH 5.5, 50 mM KCl, incubated 40 min at room temperature with increasing amounts of recombinant hnRNP A1 (0–12 μg). Lane (Δ,A1) indicates the i M incubated 40 min at room temperature, with denatured hnRNPA1 in binding buffer (see Methods); ( C ) EMSA at pH 5.5 of hras -1 Y with BSA or denatured hnRNP A1 and EMSA of hras -1 Y (m) with hnRNP A1; ss = single-stranded oligonucleotide; 1:1 and 1:2 DNA-protein complexes.
Article Snippet: Radiolabelled oligonucleotides (10 nM) were incubated for 30 min at 20 °C with increasing amounts of hnRNP A1 (0–12 μg) as specified in , in 50 mM Tris–acetate, pH 5.5, 50 mM KCl, 1 mM DTT, 8% glycerol, 1% Phosphatase Inhibitor Cocktail I (Sigma, Milan, Italy), 5 mM NaF, 1 mM Na3 VO4 , 2.5 ng/μl salmon sperm DNA (binding buffer).
Techniques: Chromatin Immunoprecipitation, Sequencing, Positive Control, Negative Control, Incubation, Recombinant, Binding Assay