RT-PCR analysis
Adult C57BL/6 male mice were used in this study. Total RNAs purified from the cerebellum, spinal cord, sciatic nerve, DRG and cultured Schwann cells using TRIzol (Invitrogen Corp.) and RNeasy kit (Qiagen GmbH) were analysed by RT-PCR using SuperScript II and random primer (Invitrogen Corp.), and PCR amplification (40 cycles; 94°C-30 s, 60°C-30 s and 68°C-5 min) with Expand Hi-Fidelity DNA polymerase (Roche Diagnostics) and ADAM22-specific primers. To detect splicing variants in the cytoplasmic domain, a forward primer was placed just upstream of the transmembrane domain and a reverse primer was set on the terminating exon. The primers used in this study were as follows. ISH04-forward: 5'-AACAGGCACTGGACAGGGGCTGAC-3' and ISH04-reverse: 5'-AATGGATGTCTCCCATAGCCTGGC-3'.