Canine Mammary PAD2 Expression Figure 4. PAD2 association with histone citrullination. (A) PAD2 is detected in the nucleus and cytoplasm of mammary epithelial. PAD2 expression is shown in green and nuclear DAPI stain is in blue with white arrows denoting punctuate PAD2 staining in euchromatic nuclear regions (10006). (B) PADs target histones from mammary gland epithelial cells for citrullination and this activity is restricted to late diestrus. Canine mammary tissue samples from anestrus, estrus/early diestrus and late diestrus were examined by SDS-PAGE and probed with an anti-pan-citrulline antibody. (C) Late diestrus mammary tissue sections were subject to a modified IF protocol using the anti-pan-citrulline antibody. The panel shows pan-citrulline staining in green and nuclear DAPI stain in blue (10006). (D) Late diestrus canine mammary tissue samples and in vitro citrullinated CMT25 histones (as a positive control) were examined by SDS-PAGE and probed with the histone tail citrullination specific antibodies H3cit 2-8-17, H3cit 26, or H4cit 3. (E) Western blot analysis of CMT25 cells treated with EGF for 24 hours show increased histone tail citrullination using a H3 cit 2-8-17 specific antibody. Equal loading was confirmed using a mouse histone 3 antibody. doi:10.1371/journal.pone.0011768.g004 estrus through the early and mid-luteal phase thus paralleling the observed increase in PAD2 expression [23]. Additionally, we show that there is increased citrullination of CMT25 histone 3 tail arginine residues 2, 8, and 17 after EGF treatment, further supporting our hypothesis that histones are targets for PAD2. Induction of citrullination by EGF has not previously been reported and our lab is currently working to understand the up- and downstream signaling pathways that mediate this increase in PAD2 and its catalytic activity. One unexpected result from our IHC and IF experiments is that, in addition to expected cytoplasmic and peri-nuclear localization, PAD2 is also detected in the nucleus of mammary PLoS ONE | www.plosone.org 7 July 2010 | Volume 5 | Issue 7 | e11768