Biotin was esterified to the ribose ring of 8-azido-ATP similar to the method used by Schafer and de Lean with the following modifications (26, 70): A 2.5-fold molar excess of carbonyldiimidazole over biotin was used to activate the biotin carboxyl group, and 8-azido-ATP was added as a 10 mM stock in buffer as supplied by Jena Biosciences with a 22.5-fold molar excess of biotin over 8-azido-ATP. The reaction was allowed to incubate until precipitates were clarified into solution. No further reaction was observed after this point. The completed reaction was purified by high-performance liquid chromatography (HPLC) using a 1.5 cm by 15 cm column of AGMP-1 (anion exchange resin, Bio-Rad) and a concave-upward gradient of trifluoroacetic acid from 3 to 300 mM at 3 ml/min over 60 min. The eluted nucleotides were detected using a Beckman 166-UV detector set at 280 nm. The biotinylated product was eluted at 56.1 min, and was collected and dialyzed against ultrapure water in 500 MWCO dialysis tubing at 4°C to remove trifluoroacetic acid. After dialysis, samples were lyophilized and stored at −20°C until use. Unreacted 8-azido-ATP was collected (elution time, 48.1 min), dialyzed as above, and used for further rounds of synthesis and purification of the biotinylated product. By analysis of HPLC peak intensities, the reactions yielded 40% product. Membranes were prepared from transfected HEK293T cells as above and resuspended in a binding buffer consisting of 50 mM Hepes (pH 7.4), 100 mM NaCl, 50 mM NaF, 0.5 μM CNP, 20% glycerol, 2 mM MgCl2, 1 μM microcystin-LR, 1× Roche cOmplete Protease Inhibitors, and with or without 1 mM ATP. Samples were incubated at room temperature for 1 hour to allow hormone binding and competitive ATP binding, and then 100 μM 8-azido-2′/3′-biotinyl-ATP was added. Samples were vortexed briefly and incubated on ice for 10 min before cross-linking in a Stratalinker 2400 for 3 min. The samples were centrifuged at 25,000g for 15 min at 4°C to pellet the membranes, supernatants were aspirated, and pellets were solubilized in 1 ml of immunoprecipitation buffer. Solubilized samples were immunoprecipitated overnight using 20 μl of FLAG-M2-agarose beads, fractionated by SDS–PAGE (polyacrylamide gel electrophoresis), and samples were split to run on two identical 8% resolving gels. Both gels were transferred to Immobilon-FL PVDF membrane and blocked with Odyssey Blocking Buffer diluted 1:1 with PBS. One blot detected total protein abundance by blotting against the N-terminal FLAG epitope as described above, and one blot detected biotin using a 1:2000 LiCor IRDye 800 Streptavidin in blocking buffer plus 0.15% Tween 20 and 0.08% SDS.

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