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In perfusion experiments, buffer was sampled from the myograph chamber and the effluent from the artery under study.
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Following vortexing, 20 µL of CER II buffer was added per sample (from kit with the addition of half a protease and half a phosphatase tablet dissolved).
The sample buffer was prepared from 0.5 M Tris HCl buffer pH 6.8, 10%% (w/v) glycerol, 0.5 % (w/v) SDS, bromophenol blue and 5%% β-mercaptoethanol.
After 2 h hybridization at 46°C, the hybridization buffer was removed from the samples.
For all the measurements, the spectrum of the buffer was deduced from the sample.
NuPAGE SDS PAGE sample buffer was from Invitrogen (Paisley, UK).
Nupage lithium dodecylsulfate (LDS) sample buffer was from Invitrogen (Carlsbad, CA, USA).
Tissue culture reagents, Novex 4 12% Bis-Tris gels and NuPAGE LDS sample buffer was from Invitrogen.
All measurements were performed in triplicate, and the fluorescence intensity of the buffer was subtracted from that of the samples.
Lysis buffer was purchased from Cell Signaling Technology.
Titration of ligand to buffer was performed and subtracted from the sample to correct for the heat of dilution.
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