Fig 1: S839I mice display higher cGMP levels in the small intestine and diarrhea-like symptoms. (A) Transcript levels of Gucy2c, Guca2a, and Guca2b in the ileum and colon were determined by RTqPCR and normalized to the expression of Gapdh. Each dot represents an individual mouse, and values shown are the mean ± SD; P values shown are from unpaired, two-tailed t tests with Welch’s correction for each gene. Seven female mice were used for this analysis across experiments repeated twice. (B) Western blot analysis of membranes prepared from ileal and colonic epithelial cells was performed using a monoclonal antibody to GC-C, and data shown from three female mice are representative of experiments repeated thrice. The expression of GC-C was normalized to expression of villin. Each dot represents individual mice from the experiment. Values represent mean ± SD, and P values shown are from unpaired, two-tailed t tests performed for each tissue with Welch’s correction. The top band in colonic epithelial cells is a nonspecific band since it was seen in membranes prepared from GC-C knockout mice (data not shown). (C) Receptor content in IEC membranes prepared from the ileum and the colon. Each dot represents data from membranes prepared from three female mice. Experiments were repeated thrice, and data shown are from one experiment. Values represent the mean ± SD, and P values are from unpaired, two-tailed t tests with Welch’s correction. (D) Cyclic GMP levels in IECs of wild type and S839I mice. Cyclic GMP levels were estimated using ELISA. Each dot represents an individual mouse, and values represent mean ± SD with P values from unpaired, two-tailed t tests with Welch’s correction. Experiments were repeated twice, and data shown are from a single experiment with four female mice. (E) Gut transit in wild type and S839I mice, as measured by presence of carmine red in fecal pellets following oral gavage. Each value is from an individual mouse, and the bar represents the mean. Data were analyzed by an unpaired, two-tailed t test with Welch’s correction, and P values are shown. For wild type mice, data are shown from nine male and seven female mice. For S839I mice, data are from 12 male and 5 female mice across experiments performed at least thrice. (F) Estimation of bowel frequency in wild type and S839I mice. Each value shown represents data from a single mouse, and P values from an unpaired, two-tailed t test with Welch’s correction are shown. For wild type mice, data from 17 males and 15 females are shown, and from S839I mice, data from 26 male and 16 female mice are shown across experiments performed at least thrice. (G) Fecal water content in wild type and S839I mice. Fecal weight was measured before and after lyophilization. Each dot represents an individual mouse, and P values shown are from an unpaired, two-tailed t test with Welch’s correction. For wild type mice, data are from 20 male mice and 16 female mice. For S839I mice, data are from 15 male mice and 19 female mice across experiments performed at least thrice.