Analysis of critical amino acids in FaeG for APN binding. A Confocal microscopy images. We measured the critical residues of FaeG for APN binding using pEC129-APN IPEC-J2 cells and the point-mutations in the candidate FaeG peptides using confocal microscopy. B ELISA assays. We used the point-mutations of FaeG peptides to determine the critical residues in APN binding. We repeated the experiments thrice and data are expressed as mean ± standard deviations (*p < 0.05, **p < 0.01). C His pull-down assays. The APN protein is a 2-mer structure, has two bands, and is expressed at 16 °C. We studied the binding between the point-mutations of FaeG and APN proteins using the Pierce™ His protein interaction Pull-down kit. Western blotting by using anti-F4 monoclonal antiserum and anti-APN polyclonal antibodies for detection, the intensity of each band for evaluating the binding activities of APN proteins with FaeG variants.