1:10,000 dilution of anti-rabbit or anti-mouse conjugated HRP (Jackson ImmunoResearch Labs, West Grove, PA) for 2 hr at room temperature. All blots were washed for 60 min (6610 min) with TBS-Tween after secondary antibody and then visualized by chemiluminescence using Millipore Immobilon Western. To confirm equal protein loading, membranes were stripped and re- probed with anti-b-actin (Abcam ab8227) or anti-cytokeratin (Dako) diluted 1:2000. Western blots for pan-citrulline were electrophoresed and transferred according to standard protocols, however prior to blocking, membranes were subject to an overnight acid treatment at 37uC to chemically modify the citrulline residues according to the manufacturer’s protocol (Millipore). The following morning, membranes were washed in ddH2O three times and then blocked and probed with the pan- citrulline antibody (1:1000). Densitometry measurements were obtained from three independent western blots and measured using the BioRad Versdoc 4000. All treatment groups are compared to vehicle treated control in each experiment. qPCR Three independent samples of frozen canine mammary tissue from each of the three estrus cycle stages were cut in .0.5 cm sections and placed in RNAlater-Ice (Ambion, Austin, TX) for 24 hours to help stabilize the mRNA. After 24 hours, tissue was transferred to a new tube containing Qiagen RLT lysis buffer and homogenized (Qiagen, Valencia, CA). Messenger RNA was purified according to the Qiagen RNeasy protocol including on column DNAse treatment to remove genomic DNA. The resulting mRNA purified from canine mammary tissue was quantified and 0.5 mgs reverse transcribed using ABI High Capacity RNA to cDNA kit according to manufacturer’s protocol (ABI, Foster City, CA). Complementary DNA was subject to qPCR analysis with intron spanning primers specific for the 75 kDa isoform of PAD2 (FWD 59-TGAGAGCCTCGTGCAAGAGAACC-39 and REV 59-CGTGAGCCCCAACTCCTTCTTG-39). Ribosomal protein S5 (RPS5) is a validated canine reference gene and was used as our endogenous control for qPCR analysis (FWD 59-TCACTGGT- GAGAACCCCCT-39 and REV 59-CCTGATTCACACGGCG- TAG-39) [17]. Quantitative PCR data was analyzed by the delta/ delta Ct method in which all PAD2 Ct values are adjusted to corresponding RPS5 levels. For estrus cycle stage and hormone/ growth factor treatment comparisons, the anestrus or vehicle treated control samples are normalized to 1 and all values are expressed as the mean 6 SEM. Statistical Analysis All experiments were independently repeated at least three times. Values were expressed as the mean 6 the SEM. Means were separated by one-way ANOVA using Tukey’s HSD and P,0.05 was considered statistically significant. Results Canine PAD2 is expressed in the mammary gland and its expression varies in an estrous cycle-dependent manner Canine mammary tissue undergoes pronounced morphological rearrangement over the course of the estrous cycle. Although hormonal changes clearly facilitate this dramatic reorganization, the downstream mechanisms by which these hormones carry out this transformation remain unclear. To begin investigating the possibility that PAD2 may play a role in these reorganization events, we obtained mammary tissue from naturally cycling beagles. Mammary tissue sections were staged by a veterinary pathologist using morphological criteria as described Canine Mammary PAD2 Expression in a recent review article [18] and were classified as being in one of three stages: anestrus (showing involution), estrus/early diestrus (showing edema, and stromal and epithelial proliferation) or late diestrus (showing active lactation). Canine mammary tissue at anestrus displayed limited poorly differentiated nests of epithelium and this epithelium showed weak PAD2 staining (Figure 1A a–b). Interestingly, mammary tissue in estrus/early diestrus showed characteristic alveolar proliferation as confirmed with Ki67 staining (Figure 1B). PAD2 staining in this stage is sparse and primarily localized to the scattered expanding end terminal alveolar units (Figure 1A c–d, arrow). However, by diestrus when mammary ducts are well differentiated, PAD2 expression was present in all the alveolar units and this signal was not detected in either ductules or ducts (Figure 1A e–f, arrow). In stark contrast, PAD4 IHC staining of canine mammary tissue sections indicate that PAD4 staining is lowest during late diestrus supporting the hypothesis of a unique role for PAD2 during late diestrus (Figure S1B). To confirm staging of late diestrus samples and milk production, we co-stained our tissues with wheat germ agglutin (WGA), which stains the apical membrane of actively lactating mammary glands. Results showed that PAD2 expression also strongly correlates with WGA staining at diestrus, thus potentially linking PAD2 expression with lactation (Figure 1B). Finally, we used TUNEL staining to determine if any of our estrous cycle staged mammary tissue samples show high levels of apoptosis as occurs during mammary gland involution. None of our samples showed a high degree of apoptosis/necrosis indicating that the tissues were not actively involuting; however the anestrus sections did display the most apoptotic cells (data not shown). Thus, by both morpholog- ical and cellular IF labeling, PAD2 expression varies over the course of the canine estrous cycle with highest expression levels appearing in alveolar unit epithelium during diestrus. Since we demonstrate that PAD2 is expressed in mammary tissue, we next sought to define which cell types express PAD2 during the different phases of the canine estrous cycle. To accomplish this objective, we used IF microscopy on the same tissue sections utilized in the IHC experiments. To label and visualize specific cell populations within the mammary tissue we used antibodies specific for PAD2, cytokeratin (an epithelial marker), p63 (a myoepithelial marker) [19], or DAPI (nuclear stain). Regarding PAD2 tissue localization, results showed that, similar to cytokeratin, PAD2 is primarily expressed in epithelial cells. Co-staining with the anti-p63 antibody was then carried out to investigate if PAD2 primarily localized to myoepithelial or luminal epithelial cells. Results showed that PAD2 expression does not overlap with myoepithelial p63 positive cells and is thus mainly confined to mammary luminal epithelial cell populations (Figure 1C). PAD2 mRNA and protein levels are highest during canine diestrus We next examined PAD2 mRNA and protein levels from the same staged tissue extracts by qPCR and western blot in an effort to corroborate our IHC findings. Intron spanning primers specific to the catalytically active 75 kDa isoform of PAD2 were used to detect PAD2 mRNA and expression was normalized to a well characterized canine reference gene, RPS5 [17]. Messenger RNA levels were seen to significantly increase approximately 1 fold from anestrus to estrus with an additional fold increase being observed from estrus to diestrus (P,0.01) (Figure 2A). In concordance with our IHC and qPCR analysis, staged mammary tissue protein samples were examined by SDS-PAGE and confirmed that PAD2 expression is highest in late diestrus PLoS ONE | www.plosone.org 3 July 2010 | Volume 5 | Issue 7 | e11768