Fig. 5.

Phosphorylation of ORM on T403/T404 and Y398/402 determines the dynamic properties of TJs. (A,B) OCLN^WT (WT), OCLN^DM (DM), OCLN^T403/404A (T403/404A), OCLN^T403/404D (T403/404D), OCLN^Y398/402A (Y398/402A) and OCLN^Y398/402D (Y398/402D) cell monolayers were incubated with low-Ca²⁺ medium (LCM) or normal-Ca²⁺ medium (NCM) for 1–24 h. Live-cell images for EGFP fluorescence were collected before and after incubation (A). Cell monolayers fixed at 1 h and 16 h were stained for EGFP-Occludin and ZO-1 (B). Scale bars: 50 µm. (C,D) OCLN^WT, OCLN^DM, OCLN^T403/404A, OCLN^T403/404D, OCLN^Y398/402A and OCLN^Y398/402D cell monolayers on transwell inserts were incubated with LCM for 16 h and TER (C) and FITC-inulin flux (D) were measured. Values are means± s.e.m. (n=6). Asterisks indicate the values that are significantly (P<0.05) different from corresponding values for OCLN^WT cell monolayers. (E–G) FRAP analysis of EGFP was performed in OCLN^WT (WT), OCLN^DM (DM), OCLN^T403/404A (T2A), OCLN^T403/404D (T2D), OCLN^Y398/402A (Y2A) and OCLN^Y398/402D (Y2D) cell monolayers. Time-lapse images were collected before and after photobleaching of several ROIs at intercellular junctions. Fluorescence intensity was measured (E) and % mobile fraction (F) and t_1/2 values (G) were calculated. Values are means±s.e.m. (n=4). Asterisks indicate the values that are significantly (P<0.05) different from the value for OCLN^WT cell monolayers.