Rats were treated with (0.05mg/mL, 10μL intrathecal injection)

Biotinylated RGS proteins (5 nM, final assay concentration) were immobilized on Luminex LumAvidin beads and incubated with diluted compound in 50 mM HEPES, pH 8.0 at room temperature, 100 mM NaCl, 0.1% Lubrol, and 1 mM DTT, supplemented with 1% BSA. To each well of a 96-well PCR plate (Axygen, Union City, CA) Alexa Fluor 532-labeled G_o was added to a final concentration of 30 nM. This mixture was incubated for 30 min at room temperature in the dark, and then it was analyzed on a Luminex 200 flow cytometer for the bead-associated fluorescence (median value). Nonlinear regression analysis of inhibition curves was performed with Prism 5.0 (GraphPad Software Inc., San Diego CA)