Fig. 5. JAM-2 and JAM-3 associate with ZO-1. (A) A ZO-1 construct comprising PDZ domains 1 to 3 of ZO-1 (ZO-1/PDZ1-3) was generated in vitro and incubated with immobilized GST-JAM fusion (JAM-1, JAM-2, JAM-3) proteins as described in the legend to Fig. 1. GST-fusion proteins lacking the C-terminal PDZ domain binding motifs were used as controls to analyse the PDZ domain-dependence of the association (JAM-19, JAM-25, JAM-35). All three JAMs bind to ZO-1 in a PDZ domain-dependent manner. (B) Lysates derived from CMT epithelial cells were incubated with immobilized JAM fusion proteins. The resulting protein complexes were subjected to SDS-PAGE and analysed by immunoblotting with antibodies directed against ZO-1; the lane marked with `lysate' contains an aliquot of CMT lysates directly immunoblotted with ZO-1 antibodies. All three JAM molecules isolate ZO-1 from CMT lysates. (C) GST-fusion proteins containing the C-terminal cytoplasmic domains of JAM-2, JAM-3, JAM-1, ESAM, CAR, claudin-1 and claudin-5 were incubated with [³⁵S]-methionine-labelled ZO-1 constructs comprising PDZ domains 1 to 3 (ZO-1/PDZ1-3) or aa residues 6-1256 (ZO-1/6-1256) as described in the legend to Fig. 4. Besides JAMs 1 to 3, ZO-1 associates with claudin-1 and claudin-5; in addition, ZO-1 associates with CAR, probably in a PDZ-domain-independent manner.