Ecl western blotting detection reagent kit

Protein samples were resolved by SDS–PAGE on NuPAGE Novex 4–12% Bis-Tris gels (ThermoFisher Scientific) with NuPAGE MOPS SDS Running buffer (ThermoFisher Scientific), or with NuPAGE MES SDS Running buffer (ThermoFisher Scientific) to improve resolution when monitoring unattached ubiquitin chains (Supplementary Figure S4) or ubiquitylated HIF1A peptide (Figure 2C). The resolved proteins were transferred onto a nitrocellulose iBlot membrane (Invitrogen, IB301031) with the iBlot Dry Transfer System (Invitrogen, Serial No.10063176). Immunoblotting was performed with the antibodies shown in Supplementary Table S1. Detection was carried out with the ECL Western Blotting Detection Reagent kit (GE Healthcare, RPN 2106). The chemiluminescent signal was captured on Hyperfilm ECL film (Amersham, GE Healthcare, 28906837) and developed with an ECOMAX X-Ray Film Processor (Protec).

Immunoblotting was performed by separating 5 μg of the different recombinant proteins (rLlPLD1 and rLlPLD2 from L. laeta, LiSMDP1 from L. intermedia, LrSMD1 from L. reclusa, and LgDerProt1 from L. gaucho), or 5 μg of L. laeta and Sicarius venom, using a 12% SDS-PAGE gels under non-reducing conditions. Additionally, 5 μg of phospholipase A₂ (PLA₂) from bee venom (Apis mellifera) (Sigma-Aldrich Co, St Louis, MO, USA) and phospholipase C (PLC) from Bacillus cereus (Sigma-Aldrich, USA) were tested. Gels were stained with Coomassie Brilliant Blue or transferred to a nitrocellulose membrane. After transfer, the membranes were blocked for 2 h with 5% non-fat milk in TBS/0.1% Tween20 (TBS-T) and incubated for 1 h at room temperature with pooled sera from Groups 1 and 2 (1:1000 dilution) or with purified and immunoselected IgGs from both groups at 1 μg/mL. Membranes were washed six times for 10 min each with TBS-T and incubated with goat anti-human HRP-IgG antibody (1:50,000 dilution) in TBS-T for 1 h at room temperature. After another six washes with TBS-T, the membranes were developed with the ECL™ Western blotting detection reagent kit (GE Healthcare, Chicago, IL, USA).

Cells maintained under either normoxia or hypoxia (0.5% O₂) were lysed, and proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were blotted onto nitrocellulose membranes to perform a semiquantitative analysis according to Sgarbi et al. [28 (link)]. Blots of resolved proteins were incubated with both mouse monoclonal anti-HIF-1α, anti IF₁ (12 kDa), anti F₁F₀ ATPase a-subunit (54 kDa) (Abcam, Cambridge, UK) and mouse monoclonal anti-β actin (42 kDa) (Sigma-Aldrich, St. Louis, MO, USA) primary antibodies. Beta-Actin was used as an internal standard. Immunodetection of primary antibody was carried out with secondary goat anti-mouse IgGH + L antibody (Life Technologies, Carlsbad, CA, USA) labelled with horseradish peroxidase. Chemiluminescent detection of the specific proteins was performed with the ECL Western Blotting Detection Reagent Kit (GE Healthcare, Waukesha, WI, USA) using the ChemiDoc MP system equipped with ImageLab software (BioRad, Hercules, CA, USA) to perform the densitometric scanning of the relative protein intensity.