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To analyze relative Hsp70 binding to TPR proteins (FLAG-IP), first the Hsp70 signal of the control reaction ( = background Hsp70 binding) was subtracted from the Hsp70 coomassie signals of each TPR protein, and these values were then normalized to the coomassie signals of the precipitated TPR proteins.
The non-specific binding was subtracted from all other values.
Non-specific binding was subtracted from total binding to obtain specific binding.
Background binding was subtracted from the value obtained for binding to the consensus DNA sequence.
Background (mock) binding was subtracted from the signal, which was expressed as a percentage of signals present in untreated cells.
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The values for non-specific binding were subtracted from total binding values (with data expressed in terms of relative fluorescence units, RFU).
Non-specific binding obtained by incubating 1 mM L- glutamate in the binding assay was subtracted from total binding to yield specific [3H] AMPA- binding.
Control experiments performed with FITC-labeled nonspecific peptide showed only minimal background binding and was subtracted from each data point.
Non-specific binding per animal was subtracted from corresponding total samples to obtain specific binding.
In case of non-specific binding, this background was subtracted from the MFI values.
Controls included wells coated and treated with everything except αv β3 (data shown) and nonspecific binding to plastic was subtracted from the final results.
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