PSB-12062 (N-(p-Methylphenylsulfonyl)phenoxazine)

Kinase experiment:

The competition binding studies are performed in assay buffer (50 mM Tris-HCl, pH 7.4) containing 1 mM EDTA and 0.2 nM [35S]ATPγS. The incubations are started by the addition of membranes (10-15 μg) and are performed in a 250 μL final assay volume. The reactions are terminated by vacuum filtration over GF/ B glass-fiber filters using a Brandell 48-well harvester. The filters are rinsed three times with ice-cold Tris-HCl buffer (50 mM, pH 7.4). The filters are punched out and transferred to 4 mL scintillation vials. Then 2.5 mL of Ultima Gold scintillation cocktail is added, and samples are counted after 6 h for 1 min each, using a liquid scintillation counter (LSC). Nonspecific binding of [35S]ATPγS is determined using 100 μM ATP[1].

References:

[1]. Hernandez-Olmos V, et al. N-substituted phenoxazine and acridone derivatives: structure-activity relationships of potent P2X4 receptor antagonists. J Med Chem. 2012 Nov 26;55(22):9576-88.
[2]. Stokes L, et al. P2X4 Receptor Function in the Nervous System and Current Breakthroughs in Pharmacology.Front Pharmacol. 2017 May 23;8:291.