Arc 7010

[¹²⁵I]IMT, which we synthesized, uptake assay was performed as previously described (25 (link)). High-performance liquid chromatography (HPLC) was performed with a COSMOSIL 5C18-AR-II column (4.6 inner diameter [ID] mm × 150 mm; Nacalai Tesque) at a flow rate of 1 mL/min with a gradient mobile phase of 20% methanol in water with 0.1% trifluoroacetic acid (TFA) to 40% methanol in water with 0.1% TFA for 20 minutes. The column temperature was maintained at 40°C. Microdissected ARC/VMH-rich region of hypothalamus was washed twice with HBSS and subsequently incubated in HBSS at 37°C for 10 minutes in a 5% CO₂ incubator. Samples were then incubated with [¹²⁵I]IMT at 4 or 37°C for 30 minutes. Samples were treated with JPH203 (MilliporeSigma SML1892) at 30 μM. The reaction was terminated by the aspiration of buffer, followed by superficial rinsing with ice-cold HBSS containing 1 mM unlabeled l-α-methyltyrosine (MilliporeSigma 286680) at 4°C 3 times to remove extracellular [¹²⁵I]IMT. Samples were lysed using 0.1 M NaOH. The radioactivity was measured by a γ-counter (Hitachi Ltd., ARC-7010). Protein concentration was determined with a Protein Assay Bicinchoninate Kit (Nacalai Tesque).