Pdquest software v 8

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 which proteins of FhES extract bind plasminogen, they were electrotransferred from 2D gels to nitrocellulose membranes at 20 V for 30 min using a Trans-Blot SD Semi-Dry Transfer cell (Bio-Rad). Blots were blocked with 2% BSA in PBS wash buffer, for 1 h at room temperature. FhES membranes were incubated overnight at 4 °C with 10 µg mL⁻¹ of human plasminogen. Then, the blots were incubated with a sheep anti-human plasminogen IgG (Acris Antibodies) at 1:1000 dilution and with a peroxidase-conjugated donkey anti-sheep IgG (Sigma) at 1:2000 dilution. These incubations were performed at 37° C for 90 min with shaking and between each step washed three times with washing buffer for 5 min per wash. Protein bands were revealed with 4-chloro naphthol. Negative controls were also used in which the plasminogen had been omitted. Membranes were digitized with the scanner GS-800 Densitometer (Bio-Rad) using the Quantity One Software v.4.6.5 (Bio-Rad). Matching of 2-D gels with the homologous Western blot to identify plasminogen-binding proteins, the assignment of molecular weights (MW) and isoelectric points (pI) of each protein were analysed using the PDQuest Software v.8.0.1 (Bio-Rad). All assays were performed in triplicate to assess the reproducibility of the spot pattern.