We examined the ability of STP0404 to inhibit IN binding to LEDGF/p75 using another HTRF-based assay [33]. Briefly, 10 nM His-tagged IN was pre-incubated in a binding buffer (150 mm NaCl, 2 mm MgCl2, 0.1% Nonidet P-40, 1 mg/ml BSA, 25 mm Tris, pH 7.4) with the compound for 30 min at room temperature, and then 10 nM FLAG-tagged LEDGF/p75 was added to the reaction. This was followed by addition of 6.6 nM anti-His6-XL665 and 0.45 nM anti-FLAG-EuCryptate antibodies (Cisbio, Inc., Bedford, MA). After overnight incubation at 4°C, the HTRF signal was recorded and the IC50 values were calculated by OriginLab software.

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