The detailed method was described previously.28 (link) Inhibition of human p97 (25 nM monomer) was carried out in assay buffer (50 mM Tris pH 7.4, 20 mM MgCl2, 1 mM EDTA, 0.5 mM TCEP) containing 0.01% Triton X-100 and 200 μM ATP. Eight-dose titration was used to determine IC50 of each compound in blocking ATPase activity, which was determined through the addition of Biomol Green Reagent (Enzo Life Sciences, Farmingdale, NY, USA).
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