GGTI298

Kinase experiment:

The given cells are lysed with Reporter Lysis Buffer and subjected to luciferase activity assay using Luciferase Assay System in a luminometer. Relative luciferase activity is normalized to protein content.

Cell experiment:

Cells are seeded in 96-well cell culture plates and treated the next day with the agents indicated. The viable cell number is determined using the sulforhodamine B assay.

Animal experiment:

The ileal loop experiment is performed in 6-8-week-old mice by a modified rabbit ileal loop assay. Following gut sterilization, the animals are kept fasted for 24 h prior to surgery and fed only water ad libitum. Anesthesia is induced by a mixture of ketamine (35 mg/kg of body weight) and xylazine (5 mg/kg of body weight). A laparotomy is performed, and the experimental loops of 5-cm length are constricted at the terminal ileum by tying with non-absorbable silk. The following fluids are instilled in each loop by means of a tuberculin syringe fitted with a disposable needle through the ligated end of the loop as the ligatured is tightened: pure CT (1 μg; positive control), saline (negative control), CT (1 μg) + TRAM-34 (different concentrations in μM as indicated in Fig. 7), CT (1 μg) + H1152 (1 μM), and CT (1 μg) + GGTI298 (different concentrations in μM), a specific inhibitor of Rap1A. The intestine is returned to the peritoneum, and the mice are sutured and returned to their cages. After 6 h, these animals are sacrificed by cervical dislocation, and the loops are excised. The fluid from each loop is collected, and the ratio of the amount of fluid contained in the loop with respect to the length of the loop (fluid accumulation ratio in g/cm) is calculated as a reflection of the efficacy of various inhibitors.

References:

[1]. Sheikh IA, et al. The Epac1 signaling pathway regulates Cl- secretion via modulation of apical KCNN4c channels in diarrhea. J Biol Chem. 2013 Jul 12;288(28):20404-15
[2]. Chen S, et al. Dissecting the roles of DR4, DR5 and c-FLIP in the regulation of geranylgeranyltransferase I inhibition-mediated augmentation of TRAIL-induced apoptosis. Mol Cancer. 2010 Jan 29;9:23.
[3]. McGuire TF, et al. Platelet-derived growth factor receptor tyrosine phosphorylation requires protein geranylgeranylation but not farnesylation. J Biol Chem. 1996 Nov 1;271(44):27402-7.