Localization of E-cadherin, occludin, and β-catenin in re-aggregating cells after treatment with SB202190.

<p>A) MCF7 cells were treated with HRG-β1 for 24 h to allow dissociation. Then SB202190 and another dose of HRG-β1 were added. Cells were monitored by time-lapse imaging. Cells were dissociated, but after addition of SB202190 they started to re-aggregate within 10 min. B) MCF7 cells were treated with the EGF domain of HRG-β1 (100 ng/ml) for 22 h. Then SB202190 was added to the culture and incubated for the indicated time. After fixation, the indicated proteins were stained and observed by confocal microscopy. Images are the sections at around the middle of the cells. Arrows indicate where the proteins are accumulated at the plasma membrane. E-cadherin and β-catenin moved to the plasma membrane within 2 min after stimulation with HRG-β1, whereas occludin did not move to the plasma membrane until 15 min after stimulation. Although E-cadherin and β-catenin accumulated at the plasma membrane, some signal later returned to the cytosol. F-actin did not move to the plasma membrane. C) MCF7 cells were stimulated with HRG-β1 for 20 h and SB202190 was added to the medium for 2 min. The reaction was stopped by addition of a low-salt buffer and cellular fractionation was performed. E-cadherin and β-catenin were detected by Western blotting. HRG: before addition of SB202190; SB 2 m: incubation with SB202190 for 2 min. D) MCF7 cells were treated with HRG-β1 for 16 h. Then the cells were treated with SB202190 for the indicated time. Cells were lysed and the immunoprecipitation was performed using the indicated antibodies. The samples were divided into two equal parts and the levels of the indicated proteins in the immunoprecipitates were detected by Western blotting with the relevant antibodies.