Fig. 2.

Deletion of ORM enhances the association of occludin with the actin-rich fraction of cells and decreases its mobility at TJs. (A) Two days after seeding, OCLN^WT, OCLN^DM and Vec cells were fixed and stained for F-actin (F-Act), β-tubulin (β-Tub) and nucleus (Nuc). Scale bar: 50 µm. (B,C) Triton-soluble and insoluble fractions prepared from OCLN^WT, OCLN^DM and Vec cells were immunoblotted for GFP and β-actin (β-Act) (B). GFP band densities were measured and normalized to corresponding β-actin band densities (C). Values are means±s.e.m. (n=3). Asterisks indicate the values that are significantly (P<0.05) different from corresponding value for OCLN^WT cells. (D–F) FRAP analysis of EGFP was performed in OCLN^WT and OCLN^DM cell monolayers. Time-lapse images of several ROIs (regions of interest) at intercellular junctions were collected before and after photobleaching (D). Fluorescence intensity in the bleached area was measured (E) and percentage mobile fractions of OCLN^WT and OCLN^DM were calculated (F). Values in panels E and F are means±s.e.m. (n=8). Asterisks indicate the values that are significantly (P<0.05) different from the corresponding values for OCLN^WT cells.