Anti hmgb1 antibody

The release of HMGB1 in the cell culture supernatant was measured using the High Mobility Group Protein 1 ELISA kit (Cloud-Clone Corp., Houston, Texas), following the manufacturer's instructions. Results were expressed in pg/mg cell proteins, according to a titration curve previously prepared. In parallel, 20 μL of the cell culture medium were resolved by SDS-PAGE and probed with an anti-HMGB1 antibody (Sigma Chemical Co.). Blots were pre-stained with Red Ponceau, to check the equal loading of proteins.

The positive control recombinant HMGB1 (provided in dithiothreitol (DTT); R&D Systems, Minneapolis, MN) and tissue biopsies (n = 2) (12 sections, 8 μm thick) suspended in PBS were incubated with NuPAGE SDS loading buffer (Life Technologies, Carlsbad, CA) at 90°C for 15 or 25 min, respectively. Proteins were separated using a 12% NuPAGE Novex Bis-Tris gel (Life Technologies, Carlsbad, CA) and transferred onto nitrocellulose membrane using the iBlot system (Life Technologies, Carlsbad, CA). The membrane was blocked using 5% dry milk powder and 0.1% Tween-20 PBS saline. HMGB1 was detected using an anti-HMGB1 antibody (Sigma Aldrich, St. Louis, MO) in combination with a horseradish-peroxidase labeled anti-mouse antibody (GE Healthcare, Little Chalfont, UK), and an electrochemiluminescence kit (Thermo Scientific, Waltham, MA). Both the oxidized or reduced forms of HMGB1 were detected under these conditions in sizes corresponding to that reported previously (Venereau et al., 2012 (link)). It should be noted that the recombinant protein has a molecular weight of 24.9 kDa but separates as a 30–36 kDa protein in SDS-PAGE according to the manufacturer's specification.

Wound Healing Assay was assessed by scratch test as previously described (19 (link)). HT29 cells were cultured in 6-well plated at a density of 2 × 10⁵ cells/ml until confluence reached 90%. A straight-line wound was made using a 10-ul pipette tip. Cell debris and smoothed the edge of the straight-line wound were removed by a wash with PBS and cells were then maintained in a medium with a reduced percentage of FBS (1%). Then cells were exposed to cytomix [TNFα (100 ng/ml) and INFγ (250 ng/ml), Peprotech, Rocky Hill, USA] or to B-box (10 μg/ml) (HMGBiotech, Milan, Italy) in presence or absence of DPG (300 μM) or anti-HMGB1 antibody (Sigma) for 48 h. In a second set of experiments, cells were treated with cytomix for 24 h and then were exposed to DPG (300 μM, Sigma), anti-VTN (1:1,000) and anti-PLAUR (1:1,000), alone or in combination, for 24 h. In both case, cells migrated into the wounded area were visualized at 0, 6, 24, and 48 h by Hematoxylin and Eosin staining. Images of each condition were acquired at a magnification of 10X. Cellular density related to a fixed wounded area (1 mm²) was measured after 48 h using ImageJ software (available in the public domain at www.nih.gov; National Institutes of Health [NIH], Bethesda, MD, USA) (10 acquisition for each experimental point). The experiment was replicated three times.