Anti rabbit igg horseradish peroxidase

Pooled human umbilical vascular endothelial cells (HUVECs) and endothelial cell growth medium 2 (EGM2) were purchased from PromoCell (Heidelberg, Germany). Antibodies to ICAM1 and GAPDH were obtained from New England Biolabs UK Ltd. (Hertfordshire, UK). Anti-SOCS3 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). SuperSignal™ West Pico Chemiluminescent Substrate was from Fisher Scientific (Loughborough, UK). Secondary antibodies, anti-rabbit-IgG horseradish peroxidase, anti-goat-IgG horseradish peroxidase and anti-mouse-IgG horseradish peroxidase conjugates, were from Sigma-Aldrich Company Ltd. (Dorset, England). Forskolin, rolipram and N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) were obtained from Calbiochem (Paisley, UK). I942 (N-(2,4-dimethylbenzenesulfonyl)-2-(naphthalen-2-yloxy)acetamide) was purchased from MolPort (Riga, Latvia). Recombinant human interleukin 6 (IL6) protein and recombinant human soluble IL6 receptor α (sIL6Rα) proteins were purchased from R and D Systems (Abingdon, UK).

Peak 1 and peak 2 (10 μg) from the CM-Sepharose step were submitted to 12% SDS-PAGE, and the gel bands were transferred onto a nitrocellulose membrane (0.22 μm pore size) for 18 h at 30 V at 4 °C. Subsequently, the membrane was blocked with a blocking buffer (5% skimmed milk, 0.15 M NaCl, 0.05% tween-20, 0.02 M Tris-HCl, pH 7.5) for 2 h, with agitation at room temperature. Then, the membrane was washed three times with washing buffer and incubated with anti-Atr-III serum (provided by FUNED, Brazil; Atr-III: a P-III SVMP from B. atrox) (dilution 1:1000 in blocking buffer) for 2 h at room temperature. After incubation, three more washes were conducted and the membrane was incubated with anti-rabbit IgG-horseradish peroxidase (Sigma-Aldrich, diluted 1:5000 in blocking buffer) for 1 h at room temperature. Finally, after another three washes, the membrane was revealed with ECL reagent (Thermo Scientific, Waltham, MA, USA). The image was acquired using Chemidoc (Bio-Rad, Hercules, CA, USA). The uncropped Western blot is shown in the Supplementary Information.