Hnf4a and Spontaneous IBD Figure 5. Claudin-15 and ion transport are modulated in Hnf4a colon mutants. (A) qRT-PCR of claudin-4, -8 and -15 at various time points in mutants (Open bars) relative to controls (Black bars). Expression levels are shown as mean values (6 SEM) relative to controls at each time point (3–14 mice per group) and were normalized with the Tbp housekeeping gene. (B) Immunoblotting confirmed modulated protein levels of claudin-4, -8 and -15 at 12 months in mutants (n = 3, open bars) and relative controls (n = 3, black bars) normalized to stable expression of E-Cadherin (Cdh1). (C) Quantification of band intensities of each claudin (means 6 SEM; n = 3 or 4 for each group) relative to Cdh1. (D) Indirect immunofluorescence (FITC) of claudin-4, -8 and -15 in 3 month-old animals in proximal colon (representative of 4 pairs). Nuclei were counterstained with DAPI (blue) and merged with FITC (green). Scale bars, 50 mm. (E) Claudin-15 expression is lost in the upper half of the colon epithelium in mutants (Open bar) relative to controls (Black bar). (F) Colonic permeability to 51Cr-EDTA measured in colon tissues from 2 month-old mice (5 mice per group). (G) Baseline short circuit current (Isc) measured in colon tissues from 2 month-old mice (5 mice per group). Represented as mean values 6 SEM, * P#.05, ** P#.01, *** P#.001. doi:10.1371/journal.pone.0007609.g005 PLoS ONE | www.plosone.org 7 October 2009 | Volume 4 | Issue 10 | e7609