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Fig. 5. [³H]25-HC binding to Insig-2 mutants. Each assay tube, in a total volume of 100 µl of buffer A, contained 400 nM His₁₀–Insig-2–FLAG (wild-type or the indicated mutant version), 25 mM phosphocholine chloride, and the indicated concentration of [³H]25-HC (152 dpm/fmol). After incubation for 4 h at room temperature, specifically bound [³H]25-HC was measured as described in Fig. 2C. Each data point is the average of duplicate assays.