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Fig. 1. Characterization of PATJ knockdown clones. (A) Cell extracts (50 µg) from control (CT Cl 8) and PATJ KD clones (Cl 4, 5 and 12) of Caco2 cells were analyzed by SDS-PAGE and western blotting with affinity-purified antibodies against human PATJ (top panel) or aPKC (bottom panel). In clones 4 and 12, all the species of PATJ above 130 KDa were barely detectable. The asterisk indicates a band that is not affected by the siRNA used. Clone 5, in contrast to clones 4 and 12 only showed a slight decrease in PATJ expression and is also used in some experiments as a control. (B) Confocal microscopic X-Y sections at the level of tight junctions of control (CT Cl 8) and PATJ KD clones (Cl 4 and 12) of Caco2 cells. Cells grown on filters were labeled with affinity-purified antibodies against human PATJ and secondary antibodies coupled to Texas Red. Note that the monolayer in CT Cl 8 is not flat and PATJ labeling is out of the section plane chosen in some areas (white asterisks). The arrow indicates a small cluster of positive cells in Cl 12 cells. Bars, 10 µm.
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