Fig 2: Inhibition of hNHE3 by FSK requires Nedd4-2. (A) Nedd4-2 expression was stably silenced in Caco-2bbe cells by using lentiviral shNedd4-2. Scrambled shRNA (shCon) was used as a control. Expression of endogenous Nedd4-2 was determined by Western blotting using anti-Nedd4-2 antibody. Representative blots from more than 3 independent experiments are shown. (B) Caco-2bbe/hNHE3 cells stably transduced with shNedd4-2 or shCon were treated with FSK or equal volume of dimethyl sulfoxide (Con) for 30 minutes. NHE3 activity was determined as the rate of Na+-dependent pHi recovery. Results are presented as mean ± SD. n = 17. ***P < .001. (C) Caco-2bbe/hNHE3 cells transfected with pcDNA control or pcDNA/Nedd4-2-ePYmut were treated with FSK as described above. n = 20. ***P < .001 by one-way ANOVA. (D) Caco-2bbe/rbNHE3 and Caco-2bbe/hNHE3 cells transduced with shNedd4-2 or shCon were treated with 10 µmol/L FSK for 30 minutes. NHE3 was immunoprecipitated with P5D4 antibody, and ubiquitination of NHE3 was determined by using P4D1 anti-Ub antibody. The panel below shows immunoprecipitated NHE3 blotted with EM450 antibody. Knockdown of Nedd4-2 is shown in the lower panel with ß-actin as control. Representative blots from 3 independent experiments are shown. The lower graph shows the quantification of Ub normalized to NHE3 immunoprecipitated. *P < .05, ***P < .001 by two-way ANOVA Tukey post hoc test. (E) Mouse NHE3 and hNHE3 were immunoprecipitated from intestinal mucosal lysates of WT and hNHE3int mice, respectively, with EM450 anti-NHE3 antibody. Ubiquitination levels of NHE3 were determined and normalized to the amount of NHE3 immunoprecipitated. Quantification from 3 independent experiments is shown on the right. ***P < .001 by two-way ANOVA.

Fig 3: Inhibition of NHE3 by EPEC is greater for hNHE3. (A) NHE3 activity is not altered by the commensal EFC-1 strain. NHE3 activity was determined in Caco-2bbe/hNHE3 and Caco-2bbe/rnNHE3 cells treated with EPC or LB medium (control) for 90 minutes. n = 10. (B-C) NHE3 activity was determined in cells treated with EPEC or EFC-1 (Con) for 90 minutes. n = 20. ***P < .001. (D) Caco-2bbe/hNHE3 cells transfected with pcDNA control or pcDNA/Nedd4-2-ePYmut were treated with EPEC as described above. n = 20. ***P < .001 by one-way ANOVA. (E) Ubiquitination of hNHE3 was determined in Caco-2bbe cells treated with EPEC. hNHE3 was immunoprecipitated and blotted with anti-Ub antibody P5D1. Lower panel shows immunoprecipitated hNHE3. Graph on the right represents quantification of Ub/hNHE3. **P < .01. Representative of 2 independent experiments with 3 mice per group. (F) Activity of hNHE3 in Caco-2bbe cells was determined in presence of pan-PKC inhibitor (PKCi) Gö6983 or PKG inhibitor (PKGi) AP-C5. n = 12–15. ***P < .001. Statistical analysis was performed by two-way ANOVA with Tukey post hoc test.

Fig 4: FSK increases the interaction of Nedd4-2 with hNHE3. (A) Binding of GST-tagged WW domains (WW1-4) of Nedd4-2 with hNHE3 was determined by pull-down assay. n = 3. (B) Subcellular localization of HA-Nedd4-2 and hNHE3 in Caco2bbe/hNHE3 cells after 5- or 10-minute FSK treatment was determined. Representative confocal cross-sectional views of hNHE3 (red), Nedd4-2 (green), and the apical membrane marker, wheat germ agglutinin (WGA; white), are shown. Scale bar: 10 µm. Results are representative of 3 independent experiments with triplicates of each experiment. Graph on the right represents Pearson’s correlation coefficient of NHE3, and Nedd4-2 localization from more than 25 independent visual fields is shown. **P < .01, ***P < .001 vs 0 minutes. (C) Caco-2bbe and SK-CO15 cells were treated with 10 µmol/L FSK for the indicated time, and phosphorylation of Nedd4-2 at Ser342 and S448 was determined using phosphorylation specific antibodies. Representative blots from 5 independent experiments are shown. **P < .01, ***P < .001 vs 0 minutes. (D) Caco-2bbe/hNHE3 cells transfected with HA-Nedd4-2 were treated with FSK. hNHE3 was immunoprecipitated, and co-immunoprecipitated HA-Nedd4-2 was detected using anti-pS342 antibody and anti-HA antibody. Bottom panel shows immunoprecipitated hNHE3 identified using EM450 antibody. Representative results from 4 independent experiments are shown. *P < .05 vs 0 minutes.

Fig 5: The effects of CsA co-treatment with perampanel (PER) and GYKI 52466 (GYKI) on expression/phosphorylation levels of ERK1/2, SGK1, NEDD4-2 and AKT, and ubiquitination of NEDD4-2 and GRIA1. (A) Representative images for Western blot of ERK1/2, SGK1, NEDD4-2 and AKT and ubiquitination of NEDD4-2 and GRIA1 in the hippocampal tissues. (B–E) Quantifications of protein level, phosphorylation level and phosphorylation ratio of ERK1/2 (B), SGK1 (C), NEDD4-2 (D) and AKT (E) in the hippocampal tissues. (F–H) Quantifications of GRIA1 level (F) and the bindings of NEDD4-2 (G) and GRIA1 (H) with ubiquitin (Ub) in the hippocampal tissues. Open circles indicate each individual value. Horizontal bars indicate mean value. Error bars indicate SEM (*,# p < 0.05 vs. control and vehicle (Veh)-treated animals, respectively; one-way ANOVA with post hoc Bonferroni’s multiple comparison).