Haptoglobin

Serum was diluted in PBS and heated for 15 min at 90 °C before being electrophoresed on 15% SDS/acrylamide gels, followed by a transfer onto a 0.2 µm nitrocellulose membrane (BioRad, Hertfordshire, UK) for 1 h at 4 °C. Membranes were blocked for 1 h in blocking buffer (5% milk in TBS, 0.1% Tween-20) before incubating with anti-Haptoglobin (Sigma-Aldrich, Poole, UK HYB 170-06-02) diluted in block buffer overnight at 4 °C. Membranes were washed for 2 × 5 min in wash buffer (88mM Tris pH7.8, 0.1% Tween-20), prior to incubating with species-specific horse radish peroxidise conjugated goat anti-mouse secondary antibody (New England Biolabs, Hertfordshire, UK) for 1 h at room temperature. Membranes were washed (3 × 5 min) in wash buffer, and immunoreactive proteins were visualised using enhanced chemiluminesence in the BioRad Universal Hood II with Quantity One Software. Haptoglobin isolated from human plasma (Sigma-Aldrich, Poole, UK) was used as a reference, and protein molecular weights were determined using Precision Plus Standards (BioRad, Hertfordshire, UK).

Antibodies, phospho (p)-Lyn, p-PI3K, p-Akt (S473), p-ERK, and Lyn, PI3K, Akt and ERK were purchased from Cell Signaling (Beverly, MA, USA). Goat anti-rabbit/mouse IgG conjugated with peroxidase were purchased from Pierce (Rockford, IL, USA). The fluorescence antibodies, anti-human P-selectin FITC (R&D systems, Minneapolis), anti-human CD41a PE, PAC1 FITC and Annexin V FITC (BD Pharmingen™ (BD Biosciences), were purchased. Anti-hemoglobin α was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The synthetic peptides (designed from N-terminal region of GP1bα) including AA1-50 (mpllllllllpsplhphpicwvskvashlevncdkrnltalppdlpkdtt) and scrambled control peptide (sctvrltknhdadedtlalppklnmvlplseilchlplvpsklplhllpp) were purchased from (GL BioChem, Shanghai). These peptides have been used successfully in our recent studies [13 (link), 14 ]. Majority of other laboratory chemicals including hemoglobin S (HbS with a purity of 98.5% isolated through Sephadex G-25 column (gel filtration chromatography) followed by ion exchange using CM-sepharose fast flow) were purchased from Sigma-Aldrich, St. Louis, USA. Hemopexin was purchased from Prospec (Prospec, Ness-Ziona, Israel), haptoglobin (Sigma Aldrich, St. Louis, USA) and ADP Colorimetric Assay kit was purchased from BioVision (BioVision,CA, USA).

Endotoxin-free, disulfide HMGB1 was kindly provided by Dr. H. Yang (Feinstein Institute for Medical Research, NY) or purchased from HMGBiotech (Milan, Italy). Minocycline (glial inhibitor), alpha-1-antitrypsin (A1AT, protease inhibitor), haptoglobin (sequesters HMGB1) and sivelestat (neutrophil elastase inhibitor) were all purchased from Sigma-Aldrich (St. Louis, MO). Intrathecal (i.t.) delivery was performed in animals deeply anesthetized with 4% isoflurane and maintained on 2.5% isoflurane during the lumbar puncture procedure using a 30-gauge needle inserted into the space between L4-L5 vertebrae. Tail flick was noted as an indicator for a successful i.t injection. Animals were injected i.t with 1 μg disulfide HMGB1 or phosphate buffered saline (PBS) as vehicle control. In pharmacological experiments, 1 μg disulfide HMGB1 was injected i.t. either alone or in combination with 30 μg Minocycline, 15 μg haptoglobin, 15 ng A1AT, or 0.5 ng sivelestat. The following day, the animals were given i.t. injection of either 30 μg Minocycline, 30 ng A1AT, 1 ng sivelestat, or vehicle (PBS). All reagents were injected in a total volume of 5 μL i.t.

Organic solvents of HPLC grade,
sodium chloride, hydrogen chloride, piperazine, bis-tris-propane,
and 1,4-dimethylpiperazine at highest available purity were purchased
from Sigma-Aldrich GmbH (Taufkirchen, Germany). Ribonucleases from
bovine pancreas (CAS-No 9001-99-4)—A with purity above 90%
and B with purity above 80%—as well as purified proteins from
human plasma (HSA, IgG, alpha-1-antitrypsin, haptoglobin, alpha-1-acid
glycoprotein) were also purchased from Sigma-Aldrich GmbH at the highest
available purity. Alpha-1-acid glycoprotein from bovine plasma with
99% purity was purchased from the same vendor.
Phosphate-buffered
saline (10× PBS), pH 6.6, was prepared in-house. Igepal-CA630,
dithiothreitol, 2-methylpiridine borane complex, procainamide hydrochloride
(ProA), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich,
St. Louis, Missouri, USA. Glacial acetic acid was produced by Merck,
Darmstadt, Germany; acetonitrile (ACN, LC-MS grade) was from Honeywell,
USA; sodium dodecyl sulfate (SDS) was from Invitrogen, Carlsbad, California,
USA. Peptide-N-glycosidase F (PNGase F) was from
Promega, Madison, Wisconsin, USA.

Protein extract (20 µg), obtained as described above, was resolved by SDS-PAGE (10%) and transferred to an NM (Bio-Rad 2 µm). After blocking (2% BSA in TBS-T buffer) of the free sites, the membrane was incubated (overnight at 4 °C) under agitation with a solution of 10 µg/mL of human adult hemoglobin (Sigma-Aldrich, Burlington, MA, USA), followed by a new incubation with anti-Hb antibody (1:5.000; Sigma-Aldrich, H4890). The antigen-antibody complex was revealed by chemiluminescence. Haptoglobin (Sigma-Merck, Burlington, MA, USA) was used as a control.