Short Communication Immunohistochemical localisation of 14-3-3 r protein in normal canine tissues Alejandro Suárez-Bonnet a, * , Juana Martín de las Mulas b , Pedro Herráez a , Francisco Rodríguez a , Antonio Espinosa de los Monteros a a Unit of Histology and Animal Pathology, Institute for Animal Health, Veterinary School, University of Las Palmas de Gran Canaria, Trasmontaña s/n, 35413 Arucas (Gran Canaria), Spain b Department of Comparative Pathology, Veterinary School, University of Córdoba, Campus de Rabanales, 14014 Córdoba, Spain article info Article history: Accepted 12 May 2009 Keywords: 14-3-3 r Canine Epithelial marker Immunohistochemistry Stratifin abstract The 14-3-3 r protein, also called stratifin, belongs to the highly conserved family of 14-3-3 acid proteins, which are involved in the modulation of diverse signal transduction pathways. Loss of 14-3-3 r expres- sion has been observed in several types of human cancers, suggesting that it may have a role as a tumour suppressor gene. The 14-3-3 r protein has been localised in normal human tissues exclusively in various epithelial cell types. The aim of the study was to investigate the expression and the distribution pattern of 14-3-3 r by immunohistochemical analysis in normal canine tissues. Immunohistochemical expression of 14-3-3 r was demonstrated in several normal canine tissues with some minor differences of distribu- tion pattern compared with human tissues. It appears that 14-3-3 r is a very specific epithelial cell mar- ker in normal canine tissues. Ó 2009 Elsevier Ltd. All rights reserved. The 14-3-3 proteins are members of a family of highly con- served, small, acid polypeptides of 28–33 kDa that are found in all eukaryotic species (Hermeking, 2003). They have a crucial role in a wide variety of cellular responses, including cell cycle progres- sion, DNA damage checkpoints and apoptosis (Hermeking, 2003). Of the seven isoforms, 14-3-3 r, also called stratifin, has been most directly linked to tumour development (Hermeking, 2003; Lodygin and Hermeking, 2006). It is a protein kinase-dependent activator of tyrosine and tryptophan hydroxylase and endogenous inhibitor of protein kinase C (Ichimura et al., 1988). The 14-3-3 r protein is a cell regulator and causes G2/M arrest by sequestering the cdc2–cyclin B1 complex in the cytoplasm and preventing its nuclear localisation, which is required for progres- sion through mitosis (Chan et al., 1999). Furthermore, 14-3-3 r has been shown to bind to G1-specific cyclin-dependent kinase 2 (cdk2), suggesting that it may also regulate G1/S progression (Lar- onga et al., 2000). Loss of 14-3-3 r expression results in malignant transformation in vitro and supports tumour formation in vivo, suggesting a role as a tumour suppressor gene (Hermeking, 2003; Lodygin and Hermeking, 2006). In spite of the ubiquitous distribution of most of the 14-3-3 iso- forms, the expression of 14-3-3 r is restricted to epithelial cells and increases during epithelial differentiation (Hermeking, 2003). These findings prompted us to analyse the distribution of 14-3-3 r protein in normal canine tissues. So far and to the best of our knowledge, no previous assessment of 14-3-3 r immunoexpres- sion in canine tissues has been reported. Normal canine tissues were obtained from four necropsy cases (two males, two females). Both males and one female dog were euthanased because of traffic accidents and had multiple fractures, and the second female died during recovery following caesarean surgery. For the immunohistochemical study, tissue sections were de- waxed in xylene, taken through ethanol, blocked for endogenous peroxidase in methanol (10 min), and subjected to a high-temper- ature antigen retrieval technique (3 min of pressure cooking in citrate buffer pH 6.0). A goat polyclonal antibody specific for the N-terminus of the 14-3-3 r isoform (N-14; Santa Cruz Biotechnol- ogy; 1:60; 16 h at 4 °C) and the avidin–biotin–peroxidase complex (Vector Laboratories) were employed (Simpson et al., 2004). Human skin was used as positive control, whereas negative controls were obtained by omitting the primary antibody. The immunohistochemical results are summarised in Table 1. 14-3-3 r immunoreactive cells were found in different locations including the epidermis (Fig. 1a), sebaceous glands (Fig. 1b), sali- vary gland (Fig. 1c), oesophagus (Fig. 1d), gall bladder (Fig. 2a), uri- nary bladder (Fig. 2b), prostate gland (Fig. 2c), mammary gland (Fig. 2d and e) and pancreatic islets (Fig. 2f). The expression was re- stricted to cells of epithelial origin. The 14-3-3 protein family acts as adaptors or ‘chaperone mole- cules’, which are able to move freely from the cytoplasm to the nu- cleus and vice versa, showing the dynamic role of these proteins in normal cells (Muslin et al., 1996). So far, the most common func- tion of 14-3-3 proteins appears to be sequestration of proteins in 1090-0233/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2009.05.014 * Corresponding author. Tel.: +34 928 45 10 95; fax: +34 928 45 11 41. E-mail address: asuarez@becarios.ulpgc.es (A. Suárez-Bonnet). The Veterinary Journal 185 (2010) 218–221 Contents lists available at ScienceDirect The Veterinary Journal journal homepage: www.elsevier.com/locate/tvjl