Anti par2

ECFCs were grown on cover slips to 60–70% confluence and fixed/permeabilized in ice-cold methanol for 10 min. Following fixation, cells were washed in PBS and blocked with 1% bovine serum albumin (BSA) for 30 min. Subsequently, the slides were incubated with anti-PAR1, anti-PAR2, VE-cadherin (Santa Cruz Biotechnology, Santa Cruz US) or VWF primary antibody (Dako, Glostrup, Denmark) (1∶50) for 1 h at room temperature. After three washing steps, slides were then incubated with Alexa Fluor 488 Rabbit Anti-Mouse and Alexa Fluor 546 Donkey Anti-Goat IgG (1∶200) (Life Technologies, Carlsbad, US) for 1 h and 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, Poole UK) (1∶100). Incubation with secondary antibodies and DAPI alone was used as a negative control. Cells were mounted in Vectashield HardSet Mounting Medium (Vector Laboratories, Peterborough UK) and examined on a LSM 510 META confocal microscope (Carl Zeiss AG, Jena, Germany).

Paraffin embedded slides derived from PAR₂ induced mammary gland tumors were deparaffinized and incubated in 3% H₂O₂. Antigen retrieval was carried out by heating (20 min) in a microwave oven in 10 mM Tris buffer containing 1 mM EDTA. After blocking the slides were incubated with the following primary antibodies: anti LRP6 (H-300 rabbit polyclonal sc15399, Santa Cruz Biotechnology, Inc. Dallas Texas, USA), anti-PAR₂ (4μg/ml [SAM11] Santa Cruz), Cy2 conjugated anti-rabbit IgG, and Cy3-conjugated anti-mouse IgG antibodies (4μg/ml, Jackson Laboratories) were used as secondary antibodies. Nuclear staining was performed using DRAQ5 (4μM, Cell Signaling). Images were obtained using a Zeiss LSM 5 confocal microscope and analyzed with Zen software (Zeiss).

Unless stated otherwise, trypsin, purified LPS (obtained through phenol extraction) from Escherichia coli (serotype O26:B6), salts, buffers, and all other chemicals of reagent grade were purchased from Sigma-Aldrich (St. Louis, MO, USA). The specific PAR-1 agonist (TRAP6), PAR-2 agonist (AC 55541), PAR-4 agonist (AY-NH2) and the selective PAR-2 antagonist (FSLLRY-NH2) were purchased from Tocris Bioscience (Bristol, UK). Antibody-directed phosphorylated ERK was purchased from Novus (St. Charles, MO, USA), and anti-ERK was purchased from BD (Franklin Lakes, NJ, USA). Antiphosphorylated p38, anti-p38, and anti-c-JUN N-terminal kinase (JNK) were purchased from Calbiochem (San Diego, CA, USA). Anti-MCP-1 was purchased from Sigma-Aldrich. Monoclonal antiphosphorylated JNK, anti-PAR-2 (Additional file 1: Figure S1), and anti-β-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).