Cyclic adp ribose

Recombinant RCD1-His and GST-RCD1ΔWWE proteins were coupled to a Biacore CM5 sensor chip (GE Healthcare) via amino-groups. PAR (625 nM) (Trevigen) was profiled at a flow rate of 30 mL/min for 300 s, followed by 600 s flow of wash buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Surfactant P20). Mono ADP-ribose (Sigma) and cyclic ADP-ribose (Sigma) were profiled at 1 mM concentration. After analysis in BiaEvalution (Biacore, GE Healthcare), the normalized resonance units were plotted over time with the assumption of one-to-one binding.

WT and Ager^−/− male mice were anaesthetised with pentobarbital sodium (50 mg/kg). The entire bilateral hypothalami were obtained and placed separately in different wells of a 12-well plate with 0.4 ml normal Locke’s solution (pH 7.25) containing 140 mM NaCl, 5 mM KCl, 1.2 mM MgCl₂, 2.2 mM CaCl₂, 10 mM glucose, 10 mM HEPES, and 0.01% BSA, in a 35 °C water bath. The incubation medium was replaced 10 times every 3 min, as described^{80 (link)}. Beginning at the 11th replacement, the temperature was shifted to 38.5 °C. From the 13th–15th replacements, cyclic ADP-ribose (a final concentration of 100 μM, Sigma) was added into the Locke’s medium at 38.5 °C. Aliquots from the 8th replacement were preserved at −80 °C for assaying the oxytocin concentrations released from the hypothalamus.