Nuclear extraction kit

Nuclear extracts were prepared as previously described [3 –7 (link)] using the Panomics Nuclear Extraction kit (no. AY2002, Panomics/Affymetrix, Freemont, CA). GATA DNA binding activity in nuclear protein extracts was analyzed by electrophoretic mobility shift assay (EMSA) as previously described [1 (link)] using double-stranded consensus GATA-specific oligonucleotides (sense, 5′-TCGCTGGACTGATAACTTTAAAAG-3′) from the ANP promoter. Double-stranded mutant oligonucleotides (sense, 5′-TCGCTGGACTGGTAACTTTAAAAG-3′) served as controls. Lamin A/C (nuclear) served as a loading control. The formation of GATA4 protein-DNA complexes was also analyzed by ELISA (TransAM® TF ELISA kits, #46966; Active Motif, Carlsbad, CA) using equal amounts of nuclear protein extracts.

Nuclear proteins were prepared from ECs transfected with siRNA or an overexpression plasmid by using a nuclear extraction kit (Panomics, USA) following the manufacturer’s instructions. TranSignal Protein/DNA Arrays I (Panomics, USA), which identify sequence-specific DNA-binding property of 54 transcription factors (TFs), were used according to the manufacturer’s directions. In general, nuclear extracts from ECs were incubated with TranSignal probe mix (Panomics) containing 54 biotin-labeled double-stranded DNA oligonucleotides for 30 min at 15 °C. The biotin-labeled oligonucleotides that were specifically bound to the TFs were eluted and hybridized to a TranSignal array membrane containing oligonucleotides. The blots were then washed and incubated with horseradish peroxidase (HRP)-conjugated streptavidin according to the manufacturer’s instructions. Hybridization signals on the array were detected using standard chemiluminescence procedures with Hyperfilm ECL (2–10 min). Relative spot intensities were determined using ScanAlyze software to obtain numerical data for comparisons. All changes that were more than 2-fold or less than 0.5-fold were considered significant [16 (link)].