Skin samples of the injected areas were biopsied under local anesthesia

12 and 24 h after injection. All of the animal experiments were approved by the animal research committee at Tokyo University of Agriculture and Technology. Formalin-fixed, paraffin-embedded skin samples were subjected to histopathological analysis. Frozen skin samples were subjected to immunofluorescence analysis for Dsg1 and Dsg3 using a human pemphigus foliaceus serum containing anti-Dsg1 IgG (Amagai et al., 1994, 1999; Ishii et al., 1997) and an AK15 mouse monoclonal antibody against Dsg3 (Tsunoda et al., 2003) (kind gifts from Dr Masayuki Amagai, Keio University School of Medicine, Tokyo, Japan), Ulixertinib respectively. The anti-Dsg1 IgG serum and the AK15 monoclonal antibody were detected with fluorescein isothiocyanate-conjugated goat anti-human IgG (MP Biomedicals, Solon, OH) and Alexa Fluor 546 goat anti-mouse IgG (Invitrogen Corp., Carlsbad, CA), respectively. Secreted forms of the recombinant proteins representing the entire extracellular domain of canine Dsg1 (cDsg1) and Dsg3 (cDsg3), fused with the hinge region of human IgG1, an E tag and a His tag at their carboxyl termini, were produced using the baculovirus-expression

system as described previously (Nishifuji et al., 2003a, b). Five insect cell DAPT nmr supernatants containing recombinant cDsg1 or cDsg3 were mixed with 10 μg of purified new ORF protein or PBS alone, and incubated at 37 °C for 12 h. Immunoblotting Ribonuclease T1 with rabbit anti-E-tag polyclonal antibody (Bethyl Laboratories, Montgomery, TX) was performed to detect intact and/or degraded cDsg proteins. PCR was performed with the primers 5′-gcggcatgcctaaaacatatgatgaagccgaa-3′ (forward primer) and 5′-tctctatttacattcagagag-3′ or 5′-tctggatccatcttctgattcagctctttttttcaaa-3′ (reverse primers) to amplify two partial regions of the orf gene. The PCR products were resolved by electrophoresis through a 1.2% (w/v) agarose gel,

and visualized by the application of the SYBR safe DNA gel stain (Invitrogen Corp.). Nucleotide sequence data obtained in this study are available in the DDBJ, EMBL and GenBank nucleotide sequence databases under accession number AB569087. During genome sequencing analysis of S. pseudintermedius strain MS5134, an orf with significant homology to a previously reported ET gene was identified. This orf consisted of 843 bp and was predicted to encode a protein of 279 amino acid residues, including a putative signal peptide in the first 32 amino acids (Fig. 1). The mature protein derived from an orf consisting of 247 amino acid residues with an N-terminal sequence beginning with KTYDEAEIIKK, and a predicted molecular weight and pI of 26.9 kDa and 5.86, respectively. The deduced amino acid sequence of the orf was compared with previously isolated ETs including S. aureus ETs (ETA, ETB and ETD), S.