Figure 4. P0 is a dominant Aire-regulated PNS antigen in mice.

A) Indirect immunofluorescence of sciatic nerve sections using sera from either NOD.WT (WT) or NOD.Aire^GW/+ (Aire^GW/+) mice. Representative staining pattern shown of 3 independent experiments. B) Multiscreen immunoblot of whole sciatic nerve extract using sera from 4 NOD.WT (left) and 4 neuropathic NOD.Aire^GW/+ (right) mice at 15-25 weeks of age. Each lane corresponds to serum from an individual mouse. Arrow points to 25-30 kD bands predominantly recognized by 4 individual NOD.Aire^GW/+ mice. Blot shown is representative of 5 experiments. C) Amino acid sequence of P0 protein, with sequences identified by mass spectrometry underlined and bolded. D) Competition blot in which serum from a NOD.Aire^GW/+ mouse was preincubated with recombinant P0 fused with an MBP tag (left panel) or with the MBP tag alone (right panel) at increasing concentrations from 0 (left lane) to 750 (right lane) nanograms of recombinant protein. Data is representative of 2 independent experiments. E) P0 ELISA using sera from either NOD.WT or NOD.Aire^GW/+ mice. Each circle represents an individual mouse. Asterix (*) indicates significant difference between the two groups. Data shown is cumulative of 3 independent experiments, with each well done at least in duplicate. F) Real-Time RT-PCR assay for relative transcriptional expression of Ins2, GAD67, and P0 in sorted mTECs from NOD.WT and NOD.Aire^GW/+ mice. Data is normalized to cyclophilin A and relative to NOD.WT. Representative data from at least 2 independent experiments assayed in duplicate. G) Proliferation assay using ³H thymidine incorporation of splenocytes from NOD.WT and NOD.Aire^GW/+ mice pulsed with irrelevant HEL peptide (left) or P0 180-199 peptide (right) at three different peptide concentrations. Averages with standard deviations of three independent experiments are shown. Within each experiment, three replicates for each genotype and peptide concentration were performed. Splenocytes from individual mice (not pooled) were used in each experiment. Asterix (*) indicates significant difference between the two groups. n.s.= not significant.