Fig. 1.

Identification of nAG protein as a ligand for Prod 1. (A) Yeast two hybrid assay illustrating the interaction between nAG and Prod 1. (B) Consensus Bayesian phylogenetic tree of representative members of the AG family of secreted proteins, highlighting nAG, the founder member XAG2, and the human AG2 which is upregulated in several examples of cancer. (C) Pull down assay with epitope-tagged forms of nAG and Prod 1 purified after bacterial expression. Lane 1, CTGF beads; 2, nAG beads; 3, control beads + Prod 1; 4, CTGF beads + Prod 1; 5, nAG beads + Prod 1. Note that Prod 1 is only pulled down in lane 5. (D) Secretion of nAG after transfection of Cos 7 cells. Cos 7 cells were transfected with a plasmid expressing the myc-tagged nAG, or RFP as control. The medium was analysed by Western blotting with anti myc. The central lane is the nAG transfected sample, the right is the RFP, and the left is the molecular weight markers. (E) Reaction of myc-tagged nAG at the surface of Prod 1 transfected mouse PS cells. nAG conditioned medium derived as in D was reacted at 4°C with live PS cells transfected to express Prod 1. Note the purple reaction product at the membrane junction between the two cells (arrowed). Scale bar, 50μm.