Figure 3.

Soluble JAML binds to CAR in T84 intestinal epithelial monolayers. As detailed in Materials and Methods, T84 monolayers were gently permeabilized with 0.03% Triton X-100 followed by blocking with BSA and incubated with JAML-Fc (10 μg/ml) in blocking solution containing protease inhibitors for 1 h at 37°C. Monolayers were immediately fixed with 3.7% PF followed by incubation with Alexa Fluo 488–conjugated goat anti-rabbit Fc. Monolayers incubated with Fc only served as control. Binding of JAML-Fc to T84 monolayers was also performed in the presence of GST chimeras of various epithelial CTX proteins (20 μg/ml). In the X-Z image in A and en-face X-Y image in C, incubation of T84 cells with JAML-Fc resulted in characteristic TJ staining, whereas no labeling with was observed after incubation of T84 monolayers with Fc only (X-Z image in B and X-Y image in D). The TJ-labeling pattern of JAML-Fc was blocked by coincubation with CAR-GST (F) but not by coincubation with JAM-A-GST (E). For reference, G and I represent staining of T84 monolayers with anti-CAR mAb RmcB, whereas H and J represents staining of tight junctions in T84 monolayers with anti-ZO-1 mAb. Bars, 20 μm.