Anti coli

Anti-NOV rabbit polyclonal antibody Novpep5170 (Pepceuticals Ltd), against peptide CPQNNEAFLQDLELKTSRGEI, was used to analyse expression in 6 month Nov^del3+/+ and Nov^del3−/− males and females (n = 3 for each genotype and sex). Cell proliferation and apoptosis were analysed at 2, 6 and 12 months using a rabbit polyclonal to PCNA (Abcam) and PARP p85 (Promega), (n = 4 for each genotype, sex and stage). The percentage positive cells was determined for each of four quadrants, averaged over two slides by KR blinded to genotype. Collagen I and Collagen X expression was assayed using mouse monoclonals anti-ColI (1/1000, Sigma) and anti-ColX (1/10, gift of Klaus von der Mark) (n = 4 for each genotype, sex and stage). Immunohistochemistry details in Supplementary Methods.

The expression of key EMT markers and their localization were assessed by immunofluorescence in PDAC cells grown on 12 mm-diameter rounded coverslips coated with FN, LAM, COL-I (Neuvitro Corporation, Vancouver, WA, USA), or uncoated. When they were at the desired confluence, cells were washed in phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde in PBS-containing 2% sucrose for 10 min at room temperature, post-fixed in 70% ethanol, and stored at −20 °C until use. Cells were incubated with the primary antibodies anti-E-cadherin (1:2500, Becton Dickinson, Milan, Italy), anti β-catenin (1:500, Novocastra, Newcastle upon Tyne, UK), anti-N-cadherin (1:200, Santa Cruz Biotechnology, Heidelberg, Germany), anti-COL-I (1:2000, Sigma-Aldrich, Milan, Italy), anti-vimentin (1:200, Novocastra), and anti-αSMA (1:400, Sigma-Aldrich). Secondary antibodies conjugated with Alexa 488 (1:500, Molecular Probes, Invitrogen, Waltham, MA, USA) were applied for 1 h at room temperature in PBS. Negative controls were incubated omitting the primary antibody. Finally, after incubation with 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI) (1:100,000, Sigma-Aldrich), the coverslips were mounted on glass slides using Mowiol.