citrullination of the histone H3 tail at MAGEA12, PTN, and likely other promoters, plays a role in breast cancer cell proliferation. Supporting Information Figure S1 PAD2 staining in human mammary tissue is specific. As a control, human mammary tissue sections were probed with rabbit IgG at a concentration equal to that of primary PAD2 antibody and counterstained with hematoxylin. (TIF) Figure S2 PAD4 expression levels are not altered in PAD2 knock down MCF-7 cells. Whole cell lysates from shRNA control and PAD2 knock down MCF-7 cells were analyzed by western blot using an anti-PAD4 antibody and an anti-b-actin antibody for loading control. RNA was purified from PAD2 knock down MCF-7 and shRNA control cells, reverse transcribed, and resulting cDNA used in qPCR reactions containing TaqMan assays to PAD4 and GAPDH as the reference gene control. Data represent the means 6 SEM of four independent experiments performed in triplicate. All values are normalized to shRNA control samples and bars represent the means 6 SEM. Means were separated by Student’s T-Test (P,0.01). (TIF) Figure S3 PAD2 associates with chromatin in the nucleus of MCF-7 cells. (A) PAD2 is endogenously expressed in MCF-7 cells and is detected in multiple cellular compartments. Wild type and PAD2 over-expressing MCF-7 whole cell lysates were subject to SDS-PAGE and probed with an anti-PAD2 antibody. Anti- Rabbit IgG was used as a negative control. (left panel). Endogenous MCF-7 cellular proteins were also separated into cytoplasmic, chromatin, and soluble nuclear pools by fractionation methods and examined by western blot (right panel). Cleanliness of fractionation was determined by stripping membranes and re-probing with antibodies for TBP (nuclear) and SOD4 (cytoplasmic) proteins. (TIF) References 1. Suzuki A, Yamada R, Chang X, Tokuhiro S, Sawada T, et al. 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Krishnakumar R, Gamble MJ, Frizzell KM, Berrocal JG, Kininis M, et al. (2008) Reciprocal binding of PARP-1 and histone H1 at promoters specifies transcriptional outcomes. Science 319: 819–821. PLoS ONE | www.plosone.org 11 July 2012 | Volume 7 | Issue 7 | e41242