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The chloride binding was determined using the equilibrium method proposed by Tang Luping.
Peptide binding was determined using ELISA.
Nonspecific binding was determined using 1 µM indatraline.
Unspecific binding was determined using a FITC-conjugated isotypic mouse anti-human IgG1 (DakoCytomation).
Total serum IgG binding was determined using the same second antibodies as above.
Total IgG binding was determined using the same second antibodies as described for the whole cell lysate assay above.
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Regions of HLH-1 genomic binding were determined using CARPET [8] and annotated to the nearest transcriptional start site (TSS).
Regions of Stat3 and c-Myc genomic binding were determined using Tilemap [22] and annotated to the nearest transcriptional start site (TSS) (Table S1 and S2).
In order to assess whether the biosensor is suitable for real-time monitoring of GDP formation, the kinetics of GDP binding were determined using stopped-flow.
Overrepresentation of genes with the DSX-binding was determined using a hypergeometric test compared against all genes in the Drosophila genome.
NF-κB binding activity was determined using the Light Shift Chemiluminesencent EMSA kit (Pierce, Rockford, IL).
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