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Fig. 4. E4-ORF1 blocks proper TJ localization of PATJ, PALS1 and Par3 in MDCK cells. (A) YFP-PATJ localizes at TJs of polarized MDCK-YFP-PATJ cells. Cells were fixed, permeabilized, and mounted on slides as described in the Materials and Methods. YFP-PATJ was directly visualized by fluorescence microscopy. (B) Transient expression of wt E4-ORF1 ablates proper TJ localization of YFP-PATJ, and the two proteins co-localize in punctate bodies. MDCK-YFP-PATJ cells were transfected with pCMV_Bam3-Neo-E4-ORF1 (5 µg). Each of the three panels represents the same field showing either YFP-PATJ staining, E4-ORF1 immunostaining, or their combined images (merge). Arrowheads indicate punctate bodies where YFP-PATJ and E4-ORF1 co-localize. (C) Endogenous PATJ, PALS1 and Par3 lack proper TJ localization in MDCK-E4-ORF1 cells. MDCK-E4-ORF1 cells and MDCK-IIIA cells were immunostained with PATJ, PALS1 or Par3 antibodies. Scale bars: 20 µm.

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