Recombinant apc

Animals were anaesthetised with isoflurane and oxygen. Full-thickness skin wounds extending through the panniculus carnosus were made with iris scissors, and a sterile 6 mm punch biopsy tool was used to outline a pattern on the dorsum of the mice as described before [22 (link)]. Recombinant APC (Xigris, Drotrecogin alfa (activated); Eli Lilly, Indianapolis, IN, USA) 10 µg/wound/day in a volume of 20 µL phosphate-buffered saline (PBS) or PBS alone (control) was topically applied to the wounds for 3 consecutive days. Animals were maintained under anaesthesia for 20 min to allow absorption of the solution. Wounds were left open, and animals were housed in individual cages. Wound healing was assessed by taking digital photographs and blindly measuring the wound area with a Visitrak digital device (Smith and Nephew, Macquarie Park, NSW, Australia) [22 (link)]. Mice were euthanised at indicated days and the area of skin around the healing wound was excised and processed blindly for further analysis.

Fetal bovine serum (FBS), anti-cadherin 11 antibody, and TRIzol were purchased from Invitrogen (Carlsbad, CA, USA); anti-ERK, phosphorylated ERK (P-ERK), p38, P-p38 antibodies and anti-fibroblast marker (ER-TR7), A/G Plus-agarose, scrambled control, validated EPCR, PC, and sPLA₂V small interfering RNAs (siRNAs) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); recombinant APC from Eli Lilly and Company (Indianapolis, IN, USA); RiboCellin Transfection Reagents from BioCellChallenge (Paris, France); anti-β-actin and PC/APC antibodies and recombinant TNF-α from Sigma-Aldrich (St. Louis, MO, USA); anti-human CD68 from eBioscience (San Diego, CA, USA); human IL-1β, IL-6, and IL-8 enzyme-linked immunosorbent assay (ELISA) DuoSet from R&D Systems (Minneapolis, MN, USA); anti-nuclear factor-kappa B (anti-NF-κB) p65 subunit antibody from Chemicon International (Temecula, CA, USA); recombinant sPLA₂V from Abcam (Cambridge, MA, USA); anti-human sPLA₂V antibody from Cayman (Ann Arbor, MI, USA); NE-PER nuclear extraction reagents from Pierce Chemical Co. (Rockford, IL, USA). RCR252 (EPCR blocking antibody) and RCR92 (EPCR non-blocking antibody) were kindly provided by Kenji Fukudome (Saga Medical School, Saga, Japan).