The expression of TGC-specific AI proteins by HEK293 and testicular germ cells were evaluated by Western blot analysis for detection of the presence of TGC-specific AIs antibody in EAO serum. The concentration of protein was measured with a Protein Quantification Kit-Rapit (Dojindo Molecular Technologies, Kumamoto, Japan). Samples were mixed with LDS Sample Buffer (Thermo Scientific, Rockford, IL, USA) and an equal amount of protein per lane was run on a 4–12% SDS-PAGE and transferred by iBlot (Thermo Scientific, Rockford, IL, USA). Blots were incubated with anti-FLAG antibody (Sigma-Aldrich, MO, USA) at a dilution of 1:1000, EAO serum at a dilution of 1:50, control serum (10 week mice) at a dilution of 1:50 at 4 °C overnight, followed by incubation with horseradish peroxidase-conjugated sheep anti-mouse IgG second antibody (GE Healthcare, Little Chalfont, UK) at a dilution of 1:10000 for 2 h at room temperature. The proteins were visualized by chemiluminescence using an ECL Prime western blotting detection kit (GE Healthcare, Little Chalfont, UK) according to the manufacturer’s instructions. Detection was performed with a ImageQuant 350 (GE Healthcare, Little Chalfont, UK).