GTPγS-binding assay

His-Gαi3 (100 nM) was preincubated with different concentrations of His-xDAPLE-CT (aa 1,638–1,932) for 15 min at 30°C in assay buffer (20 mM Na-Hepes, pH 8, 100 mM NaCl, 1 mM EDTA, 25 mM MgCl₂, 1 mM DTT, and 0.05% [wt/vol] C12E10]. Reactions were initiated at 30°C by adding an equal volume of assay buffer containing 1 µM [³⁵S] GTPγS (∼50 cpm/fmol). Duplicate aliquots (25 µl) were removed at different time points, and binding of radioactive nucleotide was stopped by addition of 3 ml ice-cold wash buffer (20 mm Tris-HCl, pH 8.0, 100 mm NaCl, and 25 mm MgCl₂). The quenched reactions were rapidly passed through BA-85 nitrocellulose filters (GE Healthcare) and washed with 4 ml cold wash buffer. Filters were dried and subjected to liquid scintillation counting. Background [³⁵S]GTPγS detected at 15 min in the absence of G protein was subtracted from each reaction and data expressed as percentage of the [³⁵S]GTPγS produced by His-Gαi3 in the absence of xDAPLE. Background counts were <5% of the counts detected in the presence of G proteins.