Abdominal Constriction Test in Mice were used for Pamoic acid activity evaluation. Four groups of 10 male, Swiss-Webster mice (30–35 g; Ace Animals, Inc., Boyertown, PA) were used. They were then injected with saline or one of three doses of pamoic acid disodium (25, 50, and 100 mg/kg s.c.). Twenty minutes later, each mouse was challenged with 0.6% acetic acid (0.30 ml/30 g animal i.p.) and, after an additional 5 min, was observed over the subsequent 10 min for abdominal writhing behavior.

U2OS cells transiently expressing human GPR35b and _arr2-GFP or HEK 293 cells transiently expressing mouse GPR35 and beta arr2-GFP were used 48 h after transfection. U2OS cells permanently expressing HA-GPR35a and _arr2-GFP (UGPR35_) were used for most experiments. Cells were plated onto coverslips, placed in 24-well plates, and pretreated for 1 h with 0.02 mg/ml poly-Dlysine. Cells were maintained at 37°C in 5% CO2 until ready for experiments (80–85% confluent) and washed once with HBSS before drug application and experiments were performed in HBSS. Agonist stimulated redistribution of _arr2-GFP was assessed after drug treatment for 40 min. Experiments involving antagonist were done with 15 min preincubation of antagonist for both the stable UGPR35 cells and the transiently transfected mouse GPR35 HEK293 cells. To examine reversibility of the antagonist, cells were preincubated with 100 nM CID2745687 for 10 min, then washed with HBSS five times for 5 min each before adding 1 mkM pamoic acid. Cells were then fixed with 4% paraformaldehyde for 20 min at room temperature followed by three washes with HBSS.