The APN-binding sites in the FaeG protein. A Array of overlapping FaeG peptides from the three variants (F4ab, F4ac and F4ad). Using an overlapping array of 205 peptides, we shifted three amino acids and synthesized them for the FaeG protein (Additional file 1). We detected the relative signal intensity of bound APNs to each position of the membrane by using polyclonal antibodies against the APNs. We used BSA-TBST as a negative control. Based on chemiluminescent signals we analyzed and determined the potential APN-binding sites in the FaeG. The peptide with the strongest intensity was set to 100% and all other spots were normalized to this value. We considered intensity values > 30% to be positive. B Amino acids analysis. The major APN-binding amino acids of the FaeG protein are positioned and marked with a red box. The differences of amino acids in the three serotypes are highlighted in yellow. C Confocal microscopy images. We measured how FaeG peptides interacted with APN using confocal microscopy experiments involving pEC129-APN IPEC-J2 cells and 9 overlapping peptides of FaeG (green, FITC labeled). The peptides of FaeG interacted with APN in pEC129-APN IPEC-J2 cells, the expression of APN in these cells by using polyclonal antibodies against APN and Dylight 549 goat anti-rabbit IgG secondary antibody (red), the interaction between FaeG and the pEC129-APN IPEC-J2 cell as the positive control (P) and the pcDNA™6.2-GW/miR-APN IPEC-J2 cells acted as the negative control (N). D ELISA assays. We coated APN proteins on ELISA plates, used 9 peptides of FaeG and the fusion FaeG proteins from three serotypes to determine the binding sites in FaeG. We repeated each experiment three times. The results shown are mean ± standard deviations (*p < 0.05, **p < 0.01).