multiple sclerosis [5,11]. There is also in vitro evidence that PAD2 can citrullinate arginine residues on histone H4 [12]. When incubated with histones, purified skeletal muscle PAD2 converts methyl arginine 3 histone H4 to citrulline, although no evidence yet exists to link PAD2 to histone citrullination in vivo. A complete understanding of the role of PAD2 in vivo requires the utilization of animal models to follow PAD2 expression through hormonal fluctuations across the entire estrous cycle. Compared to mice, the dog mammary gland model is more closely in line with human breast tissue from a molecular, morphological, and hormonal prospective [13]. For example, during the estrous/ menstrual cycle hormonal fluctuations in dog and human females show similar dramatic rises in E2 and progesterone (P4). Both female dogs and humans also show significant changes in mammary stromal tissue during the estrous cycle that does not occur in mice [14,15]. Additionally, the dog is a suitable model for evaluation of estrous cycle and lactation-dependant mammary changes considering dogs facilitate full lactational capabilities regardless of parturition with each cycle. This difference may be due to a species’ dependant persistent elevation of P4 throughout diestrus leading to a pseudopregnant state [14]. Therefore, here we have utilized canine mammary tissue to examine how and where PAD2 is expressed and the functional consequences of expression. In this report, we show for the first time that PAD2 is expressed in the cytoplasm and nucleus of canine mammary alveolar luminal epithelial cells. Further, we show that PAD2 expression varies across the estrous cycle, with expression initiating during mammary gland proliferation in estrus/early diestrus and increasing through lactation during diestrus. Additionally, we show that PAD2 expression at diestrus directly correlates with citrullination of the histone H3 tail in mammary epithelial cells suggesting that PAD2 may play a novel role in gene regulation in the mammary gland. Materials and Methods Materials Canine mammary tissue samples were collected from 6 dogs (2 in anestrus, 2 in estrus/early diestrus and 2 in late diestrus based on tissue morphologic changes) immediately after euthanasia in accordance with the guidelines outlined in the Report of the AVMA on Euthanasia under a Cornell University approved IACUC protocol (#2007-0073). Authors did not house the animals but collected tissues from intact female beagles between 3 and 5 years old which were being used for a different study as part of a collective effort to utilize all available tissues and decrease the number of animals used. Mammary tissue sections were snap frozen and stored at 280uC until utilized for protein and RNA experiments detailed below. At the same time, additional mammary tissue sections were placed in neutral buffered formalin and allowed to fix overnight. The following morning fixed mammary tissue was embedded, sectioned, and floated on micro-probe slides by the Cornell University Histology Core Laboratory. All canine mammary tissues were evaluated by a veterinary pathologist for stage of the estrus cycle, immunostaining and general morphology. Canine mammary tumor 25 (CMT25) cells were acquired from the Cornell University comparative oncology program and grown in phenol red free RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with charcoal stripped 10% fetal bovine serum (FBS) (Invitrogen), 1% sodium pyruvate (Invitrogen), 1% HEPES buffer (Invitrogen), 1% sodium bicarbonate (Sigma-Aldrich, St.Louis, MO) and 1% antibiotic antimycotic (Sigma-Aldrich) Canine Mammary PAD2 Expression [16]. For experiments, the cells were cultured in RPMI 1640 with 1% FBS for 24 hours and then treated with either 100 ng/ml of human epidermal growth factor (Invitrogen), 150 ng of sheep prolactin (Sigma-Aldrich) or 100 nM E2 (Sigma-Aldrich) or P4 (Sigma-Aldrich) for 24 hours and then harvested. Immunohistochemistry (IHC) and immunofluorescence (IF) IHC and IF experiments were carried out using a standard protocol. Briefly, slides were rehydrated in 3X 5 minute washes in xylene followed by single sequential 5 minute washes in 100, 95, and 75% EtOH. Slides for IHC were then incubated for 10 minutes in 0.5% hydrogen peroxide in methanol to quench endogenous peroxidases, however this step was omitted for IF slides. Next, slides were submerged in 0.01 M sodium citrate and boiled 2X for 10 minutes to retrieve antigens. After cooling, slides were washed in 1X PBS and then blocked in 10% normal goat serum and 2X casein (Vector Labs, Burlingame, CA) for 20 minutes at room temperature in a humidified microprobe chamber. Next, slides were blotted to remove excess blocking solution and then primary antibody diluted in 1X PBS was added for 2 hours at room temperature. Antibody dilutions are as follows: anti-PAD2 (ProteinTech #122100-1-AP, Chicago, IL) 1:100, anti-Cytokeratin (Dako MS3515, Carpinteria, CA) 1:100, anti-p63 (NeoMarkers MS-1084-P, Fremont, CA) 1:100, anti- Ki67 (Biogenex Labs BGX-Ki67, San Ramon, CA) 1:50, biotinylated WGA (Vector Labs, B-1025), anti-pan-citrulline (Millipore 17-347, Billerica, MA) 1:50. After washing three times, slides were incubated with a secondary antibody (biotinylated secondary for PAD2) diluted 1:200 in 1X PBS for 1 hour at room temperature then washed 3X in PBS. IHC slides were incubated in DAB chromagen (Vector Labs) solutions according to the manufacturer’s protocol, washed and then counterstained with hematoxylin and coverslip mounted. IF slides were incubated in avidin conjugated-FITC (Vector Labs), washed and then mounted using Vectashield containing DAPI (Vector Labs). IHC and IF for pan-citrulline require an additional step after antigen retrieval due to the necessity of chemical modification of citrulline residue for antibody recognition. Slides were incubated in acid solution overnight at 37uC to modify the citrulline residues according to the manufacturer’s protocol (Millipore). The following morning, slides were rinsed in ddH2O 3X before proceeding to the blocking step described above. During each experiment, duplicate slides were treated with control rabbit IgG antibody at the appropriate concentrations as a negative control. Western blotting Homogenized mammary tissue and CMT25 cells were lysed in RIPA buffer containing 20 mM Tris (pH 8.0), 137 mM NaCl, 10% glycerol, 1% NP-40, 0.1% SDS, 0.5% deoxycholate, and 0.2 mM PMSF and 1X general protease inhibitor. Protein concentration in lysates was determined by Bradford assay prior to gel loading to ensure equal protein loading. 6X sample buffer (300 mM Tris-HCl, pH 6.8, 60% glycerol, 30 mM DTT, 6% SDS) was added to yield a final concentration of 1X and lysates were boiled at 95uC for 5 min. Samples were subjected to SDS polyacrylamide gel electrophoresis on a 10 or 15% gel (acrylami- de:bis-acrylamide ratio of 29:1) and electro-blotted to Immobilin PVDF membranes (Millipore). Membranes were blocked in 1X casein diluted in Tris buffered saline (TBS). Anti-PAD2 (Protein- Tech), H3cit 2-8-17 (Abcam ab77164, Cambridge, MA), H3cit 26 (Abcam ab19847), H4cit 3 (Millipore 07-596), and Histone 3 antibody (Upstate 05-499) were diluted 1:1000 and incubated overnight at 4uC. Blots were washed and then incubated with a PLoS ONE | www.plosone.org 2 July 2010 | Volume 5 | Issue 7 | e11768