Fig. 7.

Deletion of ORM attenuates occludin mobility and Ca²⁺-depletion-mediated disruption of the AJC in the intestinal epithelium. (A,B) Total protein extracts from IEC-6 cells expressing EGFP-OCLN^WT, EGFP-OCLN^DM and EGFP vector (Vec) were immunoblotted for EGFP, occludin and β-actin (β-Act) (A). OCLN^WT, OCLN^DM and Vec cell monolayers were imaged live for EGFP (B). (C–E) FRAP analysis of EGFP was performed in OCLN^WT (WT) and OCLN^DM (DM) cell monolayers. Time-lapse images of several ROIs at intercellular junctions were collected before and after photobleaching (C). Fluorescence intensity in the bleached area was measured (D) and percentage mobile fractions of OCLN^WT and OCLN^DM were calculated (E). Values are means±s.e.m. (n=8). Asterisks indicate values that are significantly (P<0.05) different from the value for OCLN^WT cells. (F,G) OCLN^WT and OCLN^DM cell monolayers on transwell inserts were incubated in LCM, and TER (F) and FITC-inulin flux (G) were measured at various time points. Values presented in the graph are means±s.e.m. (n=6). Asterisks indicate the values that are significantly (P<0.05) different from corresponding values for the OCLN^WT group. (H,I) Fixed cell monolayers were stained for TJ proteins EGFP-occludin and ZO-1 (H) or AJ proteins E-cadherin and β-catenin (I). (J-L) Intestinal loops (∼2 cm long) from the mid small intestine of wild-type (WT) and occludin-deficient (OCLN^−/−) mice were prepared and filled with saline containing FITC-inulin with or without 1 mM EGTA. Loss of inulin from the loops was measured after 30 min incubation in DMEM (J). Values are means±s.e.m. (n=3). The asterisk indicates the value for the OCLN^−/− group that is significantly (P<0.05) different from the value for the WT group. Cryosections of the loops were immunostained for ZO-1, F-actin and nucleus (K) or E-cadherin and β-catenin (L). Scale bars: 50 µm.