Identification of prostate autoAgs by autoAb from d3tx male B6AF1 mice with EAP. (A) Cytoplasmic Ag is detected by indirect immunofluorescence in anterior prostate lobe with serum IgG autoAb from d3tx mice with EAP (i; original magnification × 200). The reaction of serum from a d3tx mouse without EAP is shown in panel ii as a negative control (original magnification × 200). Histological studies were done using an Olympus BH2 microscope (Olympus, Melville, NY), and pictures were taken with an Olympus DP12 digital microscope camera. Images were taken with 20×/__ NA (panels __) and 40×/__ NA (panels __) objectives. (B) Correlation between EAP severity and level of serum autoAb to anterior prostate extract detected by enzyme-linked immunosorbent assay (ELISA) as arbitrary units. STx indicates sham thymectomized mice. (C) Two protein bands with molecular weight of 68 kDa and 110 kDa (EAPA1 and EAPA2, respectively) in B6AF1 anterior prostate extract were immunoprecipitated by serum autoAb from d3tx mice and the aire knockout mice with EAP, detected by SDS-PAGE. (D) Prostate-specific expression of EAPA1 and EAPA2 is determined by RT-PCR. Lane 6 contains prostate RNA, whereas the lanes with negative result contain RNA of kidney, stomach, lung, brain, testis, muscle, spleen, liver, heart, pancreas, and thymus. (E) The incidence of serum autoAb that immunoprecipitated EAPA1 (□) and EAPA2 (▪) correlated with EAP severity of d3tx mice, and EAPA2 Ab is detected more frequently.