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Fig. 3. VP8 perturbs the fence function of the tight junction (TJ). In all the experiments presented here, cells were plated at confluency on Transwell inserts for 3 days. Some monolayers remained in control media, whereas others were treated for 1 hour with 4 µg/ml of GST or GST-VP8, or for 10 minutes with 1.8 mM EGTA. The membrane distribution of the fluorescent markers was determined by z-sectioning on a confocal microscope. Bar, 8 µm. (A) Bodipy®-sphingomyelin-BSA complex diffusion assay. Fluorescent lipid/BSA was loaded to the apical surface. Arrows indicate lateral membrane staining. (B) Movement of the apical membrane protein GP135 (green) to the lateral membrane. In this and the following images arrows indicate occludin (blue) staining in the TJ region of control and GST-treated monolayers. The arrowheads in GST-VP8 and EGTA-treated monolayers denote the protein that has moved to the opposite plasma membrane. (C) Movement of the basolateral membrane protein Na+K+-ATPase (green) to the apical surface. (D) Redistribution of 
ß3 integrin (green) from the basolateral to the apical surface. (E) Another set of monolayers treated in the same manner was employed for selective surface membrane labeling (AP, apical; BL, basolateral) with activated biotin. Densitometric values of ß1 integrin labeling are expressed as mean percentages of the total (apical plus basolateral)±s.e.m. Immunoblots are representative of those from three separate experiments.
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