Hormonal Functions of Cthrc1 Figure 1. Generation of Cthrc1 mice. (A) Cthrc1 gene targeting schematic showing replacement of exons 2–4 with a PGK-neo cassette. (B) Embryonic stem cell targeting with Southern blot analysis of genomic DNA from ES cell clones shows a targeted clone. (C) RT-PCR shows lack of Cthrc1 transcripts in RNA preparations of skull bone and whole brain of adult Cthrc1 mutants. doi:10.1371/journal.pone.0047142.g001 procedure were executed as previously published [7]. We conjugated the antibody to horse radish peroxidase (Pierce, ThermoFisher) following the manufacturer’s protocol and validat- ed its suitability for Western blotting using plasma samples from Cthrc1 transgenic and wild-type mice (1:2000 dilution as described [1]. Five ml of plasma were loaded per lane and immunoblotting was performed on reduced and denatured samples (Fig. 2). Validation for immunohistochemistry was performed on tissues previously shown to express Cthrc1, i.e. adventitial cells of remodeling arteries, dermal cells in skin seven days after wounding, embryonic cartilage, and absence of staining on tissue sections from Cthrc1 mice (Fig. 2). Pre-absorption of the antibody with peptide antigen was used as an additional control for specificity. Cthrc1 in Human Plasma For detection of human Cthrc1 in plasma, mouse monoclonal antibodies were raised against a synthetic peptide with sequence of the N terminus of human Cthrc1 (SEIPKGKQKAQLRQRE) using the hybridoma services of Maine Biotechnology Services (Portland, ME). Anti-Cthrc1 clones 10G7 and 19C7 detected human Cthrc1 expressed in CHO-K1 cells by indirect ELISA without amplification in the low picogram range. Protein A- purified antibodies were conjugated to magnetic beads (Pierce, ThermoFisher) following the manufacturer’s protocol. EDTA plasma was obtained from healthy volunteers. 15 ml of plasma were incubated overnight at 1uC with anti-Cthrc1 conjugated magnetic beads, and washed extensively with phosphate buffer prior to elution with 0.1 M glycine, pH = 2.6. The eluate was immunoblotted with HRP-conjugated monoclonal anti-Cthrc1 antibodies following SDS-PAGE under reducing conditions. Labeling of Cthrc1 Protein with 125I(odine) An adenovirus was generated expressing rat Cthrc1 with a C terminal myc/66His tag. CHO-K1 cells were transduced with this adenovirus and Cthrc1 protein was purified from the conditioned medium with HIS-Select affinity gel (Sigma) following the supplier’s instructions. Silver-stained SDS-PAGE gels demon- strated .95% purity of the purified protein (not shown). A BCA protein assay (Pierce) was used to determine the concentration of the purified protein. Purified Cthrc1 was labeled with 125I (Perkin Elmer) using iodination tubes (Pierce). Six mg of radioactive labeled Cthrc1 were infused into adult anesthetized Cthrc1 mice via the left carotid artery (n = 3 mice). Blood samples were obtained at indicated times and Cthrc1 levels were determined in a gamma counter. The half-life in circulation was calculated from the clearance curve. SDS-PAGE analysis followed by autoradiog- raphy was performed on 1 ml of plasma obtained thirty minutes after injection of 125I-Cthrc1 to verify its integrity. All tissues were harvested six hours after 125I-Cthrc1 injection following extensive perfusion with lactated Ringer’s solution to remove as much blood from organs as possible. 125I-Cthrc1 per mg wet weight of tissue was measured by gamma counting. Cell Culture and Western Blotting HEK293-T and CHO-K1 cells were grown as described and transfected with an expression vector for human Cthrc1 using Fugene6 HD (Roche) [4]. 48 hours after transfection the growth medium was replaced with serum-free medium and cell lysates as well as conditioned media were harvested 24 hours later for immunoblotting with HRP conjugated anti-Cthrc1 antibody. Statistical Analysis Data are expressed as means 6 standard deviation. Student’s t- test was used for all calculations. P#0.05 was considered significant. Results Generation and Characterization of the Cthrc1 Null Allele To characterize Cthrc1 function in vivo, we generated a novel Cthrc1 allele by replacing three of the four exons (exons 2–4) with a neomycin cassette (Cthrc1tm1Vli) (Fig. 1A). This mutant allele results in mice with no detectable Cthrc1 transcript in organs PLOS ONE | www.plosone.org 3 October 2012 | Volume 7 | Issue 10 | e47142