Anti fibrinogen β chain 1f9

The differential abundance of some differentially expressed proteins of interest was validated by immunoblot analyses. A first validation was performed on 3 pools of proteins, using all 15 paired samples analysed by 2D-DIGE. A second validation was performed on individual proteins extracted from cancer tissues of an additional cohort of 20 patients. Ten μg of proteins were fractionated on 12% Criterion TGX Stain-Free gels and, after gel image acquisition with the Chemidoc system (BIO-RAD) and electrotransfer onto nitrocellulose membranes. Membranes were incubated with the monoclonal antibodies anti-fibrinogen β chain [1F9] (1:500; GeneTex) and anti-β-actin (1:1000, Abcam), and the polyclonal anti-β-tubulin (1:3000, Santa-Cruz). Antibody-bound proteins were detected by enhanced chemiluminescence using the Chemidoc system after incubation with ECL HRP-conjugated secondary antibodies (1:25000 dilution, GE Heathcare) and reaction with ECL Prime Western Blotting detection reagent (GE Healthcare). The image of the gel acquired before its transfer was used as control for equal protein loading among samples.