Anti tpa

Western blot analyses were performed as described in detailed previously [23 (link)] with slightly modifications. CnAOEC and CnAOSMC previously treated with 1μg/ml of rDiACT or rDiFBAL for 24 h were lysed in ice-cold lysis buffer (20mM Tris—HCl (pH 7.5), 140mM NaCl, 10mM ethylendiaminetetraacetic acid, 10% glycerol, 1% Igepal CA-630, aprotinin, pepstatin, and leupeptin at 1μg/ml each, 1mM phenylmethylsulfonyl fluoride, and 1mM sodium orthovanadate). Non-stimulated cells were used as controls under the same conditions. Protein samples (10 μg) were separated by SDS-PAGE under reducing conditions and electrotransferred onto polyvinylidine difluoride membranes. Then, membranes were blocked before incubation with the following primary rabbit polyclonal antibodies: anti-tPA and anti-uPA (Santa Cruz Biotechnology Inc) according to the manufacturer's recommendations. After incubation with HRP-conjugated anti-rabbit secondary antibodies, bands were visualized by a luminol-based detection system with p-iodophenol enhancement. Anti-α-tubulin antibody (Oncogene Research Products) was used as control to confirm loading of comparable amount of protein in each lane. Protein expression was quantified by densitometry using the PDQuest Software v.8.0.1 (Bio-Rad).

To determine IEL and IT formation, paraffin tissue sections were stained with elastica van Gieson, as recommended by the manufacturer (Muto Pure Chemicals, Tokyo, Japan). Immunohistochemical analysis was performed as described previously [8 (link), 9 (link)]. Rabbit polyclonal anti-t-PA (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit polyclonal anti-von Willebrand factor (Dako Cytomation, Santa Clara, CA, USA), mouse monoclonal anti-α-SMA (Sigma-Aldrich, St. Louis, MO, USA), rabbit polyclonal anti-MMP2 (Novus Biologicals, Little, CO, USA) antibodies were used.

Western blot analyses were performed as previously described [21 (link)] with slight modifications. CnAOEC and CnAOSMC previously treated with 1 μg/ml of DiES for 24 h were lysed in ice-cold lysis buffer (20 mM Tris–HCl (pH 7.5), 140 mM NaCl, 10 mM ethylendiaminetetraacetic acid, 10% glycerol, 1% Igepal CA-630, aprotinin, pepstatin, and leupeptin at 1 μg/ml each, 1 mM phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate). Non-stimulated cells were used as controls under the same conditions. Protein samples (10 μg) were separated by SDS-PAGE under reducing conditions and blotted onto polyvinylidine difluoride membranes. Membranes were blocked before incubation with primary antibodies: anti-tPA, anti-uPA, anti-Annexin A2 and anti-PAI-1 (Santa Cruz Biotechnology Inc) according to the manufacturer’s recommendations. After incubation with HRP-conjugated secondary antibodies, bands were visualized by a luminol-based detection system with p-iodophenol enhancement. Anti-α-tubulin antibody (Oncogene Research Products) was used to confirm loading of comparable amount of protein in each lane. Protein expression was quantified by densitometry using Scion Image Software (Scion).